Compositions comprising a tn3 scaffold and methods of using the same

ABSTRACT

The present invention provides Tenascin-3 FnIII domain-based scaffolds that specifically bind to CD40L. The invention further provides engineered variants with increased affinity for the target. The present invention is also related to engineered scaffolds as prophylactic, diagnostic, or therapeutic agents, in particular for therapeutic uses against SLE and other autoimmune diseases and conditions.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/387,269, filed Jul. 28, 2021 (now U.S. Pat. No. 11,555,062, issuedJan. 17, 2023), which is a continuation of U.S. patent application Ser.No. 15/993,964, filed May 31, 2018 (now U.S. Pat. No. 11,104,720, issuedAug. 31, 2021), which is a divisional of U.S. patent application Ser.No. 14/347,016, filed Mar. 25, 2014 (now U.S. Pat. No. 10,000,553,issued Jun. 19, 2018), which is a U.S. National Stage Application ofInternational Application Number PCT/US2012/059477, filed Oct. 10, 2012,which claims the benefit of priority of U.S. Provisional Application61/546,028 filed on Oct. 11, 2011. Each of the above-referencedapplications are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

The contents of the electronic sequence listing(HOPA_016_04US_Sub_SeqList_ST26.xml; Size: 251,952 bytes; and Date ofCreation: Feb. 22, 2023) are herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates in general to the field of antibodymimetics, specifically to scaffolds derived from the third fibronectintype III domain of human Tenascin C useful, for example, for thegeneration of products having novel binding characteristics. Inparticular, the invention relates to CD40L-specific Tn3 scaffolds,methods of making such scaffolds, and methods of use for diagnosis andtreatment of systemic lupus erythematosus and other autoimmune and/orinflammatory disorders.

Background Art

This invention relates to CD40L-specific protein scaffolds that bind toCD40L, useful, for example, for the treatment of autoimmune and/orinflammatory disorders.

Biomolecules capable of specific binding to a desired target epitope areof great importance as therapeutics, research, and medical diagnostictools. A well known example of this class of molecules is the antibody.Antibodies can be selected that bind specifically and with affinity toalmost any structural epitope. However, classical antibodies arestructurally complex heterotetrameric molecules with are difficult toexpress in simple eukaryotic systems. As a result, most antibodies areproduced using complex and expensive mammalian cell expression systems.

Proteins having relatively defined three-dimensional structures,commonly referred to as protein scaffolds, may be used as reagents forthe design of engineered products. One particular area in which suchscaffolds are useful is the field of antibody mimetic design. Antibodymimetics, i.e., small, non-antibody protein therapeutics, capitalize onthe advantages of antibodies and antibody fragments, such as highaffinity binding of targets and low immunogenicity and toxicity, whileavoiding some of the shortfalls, such as the tendency for antibodyfragments to aggregate and be less stable than full-length IgGs.

These drawbacks can be addressed by using antibody fragments created bythe removal of parts of the antibody native fold. However, this oftencauses aggregation when amino acid residues which would normally beburied in a hydrophobic environment such as an interface betweenvariable and constant domain become exposed to the solvent. One exampleof a scaffold-based antibody mimetic is based on the structure of aFibronectin type III domain (FnIII), a domain found widely across phylaand protein classes, such as in mammalian blood and structural proteins.The design and use of FnIII scaffolds derived from the third FnIIIdomain of human tenascin C is described in PCT applicationsPCT/US2011/032184 and PCT/US2011/032188, both of which are hereinincorporated by reference in their entireties.

CD40L is a member of the TNF family of molecules which is primarilyexpressed on activated T cells (including Th0, Th1, and Th2 subtypes,and forms homotrimers similar to other members of this family. Further,CD40L has also been found expressed on Mast cells, and activatedbasophils and eosinophils. CD40L binds to the CD40 receptor (CD40R) onantigen-presenting cells (APC), which leads to many effects depending onthe target cell type. In general, CD40L plays the role of acostimulatory molecule and induces activation in APC in association withT cell receptor stimulation by MHC molecules on the APC.

Signaling through the CD40 receptor by CD40L initiates a cascade ofevents that result in the activation of the CD40 receptor-bearing cellsand optimal CD4+ T cell priming. More specifically, the cognateinteraction between CD40L and the CD40 receptor promotes thedifferentiation of B cells into antibody secreting cells and memory Bcells (Burkly, In Adv. Exp. Med. Bio., Vol. 489, D. M. Monroe, U.Hedner, M. R. Hoffman, C. Negrier, G. F. Savidge, and G. C. I. White,eds. Klower Academic/Plenum Publishers, 2001, p. 135). Additionally, theinteraction between CD40L and the CD40 receptor promotes cell-mediatedimmunity through the activation of macrophages and dendritic cells andthe generation of natural killer cells and cytotoxic T lymphocytes (seeBurkly, supra).

The interaction between CD40L and the CD40 receptor has been shown to beimportant in several experimentally induced autoimmune diseases, such ascollagen-induced arthritis, experimental allergic encephalomyelitis,oophoritis, colitis, drug-induced lupus nephritis. Specifically, it hasbeen shown that disease induction in all of these models can be blockedwith CD40L antagonists at the time of antigen administration. Theblockade of disease using anti-CD40L antagonists has also been seen inanimal models of spontaneous autoimmune disease, includinginsulin-dependent diabetes and lupus nephritis, as well as ingraft-vs-host disease, transplant, pulmonary fibrosis, andatherosclerosis disease models.

Disruption of the CD40L/CD40R pathway via CD40L blockade has been shownto be beneficial in many autoimmune mediated diseases (for example, butnot limited to systemic lupus erythematosus (SLE), rheumatoid arthritis(RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) andallograft rejection. For example, treatment with anti-CD40L antibodiesprevented or improved nephritis in a collagen-induced arthritis mousemodel (Mohan et al. J. Immuno. 154:1470). Additionally, anti-CD40Lantibodies preserved renal function in SNF1 mice with establishednephritis. (Kalled et al. J. Immuno. 160:2158). CD40L levels correlateclosely with clinical disease severity (i.e., reduction ofinflammation), and damage in target tissue in both non-humans andhumans.

SLE is a progressive and sometimes fatal autoimmune disease. The diversepresentations of lupus range from rash and arthritis through anemia andthrombocytopenia to even psychosis. There is clear evidence showing thatmany arms of the immune system are involved in the inflammatory processleading to kidney, skin, brain disease and thrombosis. Onecharacteristic feature of SLE is the loss of B cell tolerance andautoantibodies are prominent in patients with this disease. In lupuskidney disease, anti-double-stranded DNA autoantibodies can formantibody nucleosome complexes and settle in the renal glomerularbasement membrane. These immune complexes in turn activate complement,which can lead to glomerulonephritis.

Expression of CD40R as well as CD40L has been found elevated in patientswith SLE. The increased costimulatory signal likely contributes to thepathological inflammatory response found in the SLE. SLE T cells havespontaneously increased activation associated with a reduced thresholdof activation to self-antigens. Further, these cells are hyporesponsiveto further antigenic stimulation, are resistant to apoptosis, haveincreased survival after activation and have many altered intracellularsignaling pathways. Following CD40R activation on APCs by T cell CD40L,both APC and T cells become activated, produce cytokines and in SLEcontribute to the production of pathogenic autoantibodies and tissueinjury (lupus nephritis). Blockade of the CD40R/CD40L pathway iseffective, alone or in combination, in blocking disease in lupus-pronemice. In patients with SLE, a humanized anti-CD40L antibody reducedanti-dsDNA and B cells, proteinuria, and improved SLE disease severity.

However, targeting CD40L with traditional antibodies has raisedsignificant safety concerns. For example, a study with anti-CD40Lantibody 5c8 (BIOGEN®) in patients suffering with chronic refractoryidiopathic thrombocytopenic purpura (ITP) was placed on hold because ofreported thromboembolic complications (Davidson et al. Arth Rheu,43:S271). Further, additional trials with alternative antibodiesdirected against CD40L gave rise to other thrombotic relatedcomplications (Davis et al. Arth Rheu, 43:S281; Schuler,Transplantation, 77:717). Given the complications with antibody-directedantagonism of CD40L, there is an unmet need to target and antagonizeCD40L with a non-antibody alternative. Thus, targeting CD40L with aTn3-based scaffold is an attractive alternative by avoiding Fab2 and/orFc-mediated platelet aggregation and the downstream side effects.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

The invention provides a Tn3 scaffold comprising a CD40L-specificmonomer subunit, wherein the monomer subunit comprises seven betastrands designated A, B, C, D, E, F, and G, and six loop regionsdesignated AB, BC, CD, DE, EF, and FG, and wherein the Tn3 scaffoldspecifically binds to CD40L. In some embodiments, the Tn3 scaffoldcomprises a single CD40L-specific monomer subunit. In other embodiments,the Tn3 scaffold comprises two CD40L-specific monomer subunits connectedin tandem. In some specific embodiments, the Tn3 scaffold comprises twoCD40L-specific monomer subunits which are directly connected.

In some embodiments, two CD40L-specific monomer subunits are connectedby a linker. In other embodiments, the linker comprises a peptidelinker, which can be a flexible peptide linker. In some embodiments, thepeptide linker comprises a (G_(m)X)_(n) sequence wherein X is Serine(S), Alanine (A), Glycine (G), Leu (L), Isoleucine (I), or Valine (V); mand n are integer values; m is 1, 2, 3 or 4; and, n is 1, 2, 3, 4, 5, 6,or 7. In some embodiments, the peptide linker comprises SEQ ID NO: 131,SEQ ID NO: 132, SEQ ID NO: 142 or SEQ ID NO: 143.

In some embodiments, the binding of a Tn3 scaffold comprising twoCD40L-specific monomer subunits to CD40L is improved over that of a Tn3scaffold comprising a single CD40L-specific monomer subunit. In otherembodiments, the binding of a Tn3 scaffold comprising two CD40L-specificmonomer subunits to CD40L improves the action on the target over that ofa Tn3 scaffold comprising a single CD40L-specific monomer subunit. Inother embodiments, the improvement in the binding of the Tn3 scaffold toCD40L is an improvement in binding affinity, an improvement in bindingavidity, or both. In certain embodiments, the binding affinity of a Tn3scaffold comprising two CD40L-specific monomer subunits to CD40L and theTn3 scaffold protein stability are improved over those of a Tn3 scaffoldcomprising a single CD40L-specific monomer subunit. In some embodiments,the binding avidity of a Tn3 scaffold comprising two CD40L-specificmonomer subunits for CD40L and the Tn3 scaffold protein stability areimproved over those of a Tn3 scaffold comprising a single CD40L-specificmonomer subunit.

In some embodiments, at least one CD40L-specific monomer subunit in aTn3 scaffold is bound to a linker, or to a heterologous moiety. In otherembodiments, a linker or a heterologous moiety in a Tn3 scaffold isconjugated to the N-terminus or the C-terminus of a CD40L-specificmonomer subunit. In certain embodiments, the linker bound to aCD40L-specific monomer subunit in a Tn3 scaffold comprises a peptidelinker, which in some embodiments can be a flexible peptide linker. Thispeptide linker can comprise in certain embodiments a (G_(m)X)_(n)sequence wherein X is Serine (S), Alanine (A), Glycine (G), Leucine (L),Isoleucine (I), or Valine (V); m and n are integers; m is 1, 2, 3 or 4;and, n is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the peptidelinker bound to a CD40L-specific monomer subunit in a Tn3 scaffoldcomprises SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 142, or SEQ ID NO:143.

In some embodiments, the Tn3 scaffold comprises a linker which comprisesa functional moiety. In some embodiments, this functional moiety is animmunoglobulin or a fragment thereof. In certain embodiments, thisimmunoglobulin or fragment thereof comprises an Fc domain. In someembodiments, this Fc domain fails to induce at least one FcγR-mediatedeffector function. In some embodiments, this at least one FcγR-mediatedeffector function is ADCC.

In some embodiments, the Tn3 scaffold comprises at least oneCD40L-specific monomer subunit bound to a heterologous moiety. In someembodiments, this heterologous moiety comprises a composition selectedfrom the group consisting of: a protein, a peptide, a protein domain, alinker, a drug, a toxin, a cytotoxic agent, an imaging agent, aradionuclide, a radioactive compound, an organic polymer, an inorganicpolymer, a polyethylene glycol (PEG), biotin, an albumin, a human serumalbumin (HSA), a HSA FcRn binding portion, an antibody, a domain of anantibody, an antibody fragment, a single chain antibody, a domainantibody, an albumin binding domain, an enzyme, a ligand, a receptor, abinding peptide, a non-FnIII scaffold, an epitope tag, a recombinantpolypeptide polymer, a cytokine, and a combination of two or more ofsaid moieties.

In some embodiments, the Tn3 scaffold comprises at least oneCD40L-specific monomer subunit conjugated to PEG. In some embodiments,the Tn3 scaffold comprises at least one CD40L-specific monomer subunitconjugated to an albumin. In certain embodiments, this albumin is humanserum albumin (HSA). In other embodiments, this HSA is a variant HSA. Insome specific embodiments, the amino acid sequence of the variant HSA isSEQ ID NO: 133. In other embodiments, the variant HSA has at least oneimproved property compared with a native HSA or a native HSA fragment.In certain embodiments, the improved property is an altered plasmahalf-life compared with the plasma half-life of a native HSA or a nativeHSA fragment. In some embodiments, the altered plasma half-life is alonger plasma half-life compared with the plasma half-life of a nativeHSA or a native HSA fragment. In other embodiments, the altered plasmahalf-life is a shorter plasma half-life compared with the plasmahalf-life of a native HSA or a native HSA fragment.

In some embodiments, the Tn3 scaffold is fused to an HSA variantcomprising at least one amino acid substitution in HSA domain III. Inother embodiments, the Tn3 scaffold is fused to an HSA variantcomprising the sequence of full-length mature HSA (SEQ ID NO: 133 or138) or a fragment thereof, except for at least one amino acidsubstitution, numbered relative to the position in full length matureHSA, at a position selected from the group consisting of 407, 415, 463,500, 506, 508, 509, 511, 512, 515, 516, 521, 523, 524, 526, 535, 550,557, 573, 574, and 580; wherein the at least one amino acid substitutiondoes not comprise a lysine (K) to glutamic acid (E) at position 573, andwherein the Tn3 scaffold has a plasma half-life longer than the plasmahalf-life of a Tn3 scaffold not conjugated to such HSA variant.

In some embodiments, the Tn3 scaffold is fused to an HSA variant whereinat least one amino acid substitution, numbered relative to the positionin full length mature HSA, is at a position selected from the groupconsisting of 463, 508, 523, and 524, wherein said Tn3 scaffold has aplasma half-life longer than the plasma half-life of a Tn3 scaffold notconjugated to said HSA variant. In some embodiments, the HSA variantcomprises the sequence of full-length mature HSA (SEQ ID NO: 133 or 138)or a fragment thereof, except for at least one amino acid substitution,numbered relative to the position in full length mature HSA, selectedfrom the group consisting of: (a) substitution of Leucine (L) atposition 407 to Asparagine (N) or Tyrosine (Y); (b) substitution ofValine (V) at position 415 to Threonine (T); (c) substitution of Leucine(L) at position 463 to Asparagine (N); (d) substitution of Lysine (K) atposition 500 to Arginine (R); (e) substitution of Threonine (T) atposition 506 to Tyrosine (Y); (f) substitution of Threonine (T) atposition 508 to Arginine (R); (g) substitution of Phenylalanine (F) atposition 509 to Methionine (M) or Tryptophan (W); (h) substitution ofAlanine (A) at position 511 to Phenylalanine (F); (i) substitution ofAspartic Acid (D) at position 512 to Tyrosine (Y); (j) substitution ofThreonine (T) at position 515 to Glutamine (Q); (k) substitution ofLeucine (L) at position 516 to Threonine (T) or Tryptophan (W); (l)substitution of Arginine (R) at position 521 to Tryptophan (W); (m)substitution of Isoleucine (I) at position 523 to Aspartic Acid (D),Glutamic Acid (E), Glycine (G), Lysine (K), or Arginine (R); (n)substitution of Lysine (K) at position 524 to Leucine (L); (o)substitution of Glutamine (Q) at position 526 to Methionine (M); (p)substitution of Histidine (H) at position 535 to Proline (P); (q)substitution of Aspartic Acid (D) at position 550 to Glutamic Acid (E);(r) substitution of Lysine (K) at position 557 to Glycine (G); (s)substitution of Lysine (K) at position 573 to Phenylalanine (F),Histidine (H), Proline (P), Tryptophan (W), or Tyrosine (Y); (t)substitution of Lysine (K) at position 574 to Asparagine (N); (u)substitution of Glutamine (Q) at position 580 to Lysine (K); and, (v) acombination of two or more of said substitutions, wherein said Tn3scaffold has a plasma half-life longer than the plasma half-life of aTn3 scaffold not conjugated to said HSA variant.

In some embodiments, the Tn3 scaffold is fused to an HSA variantcomprising the sequence of full-length mature HSA (SEQ ID NO: 133 or138) or a fragment thereof, except for at least one amino acidsubstitution, numbered relative to the position in full length matureHSA, selected from the group consisting of: (a) substitution of Leucine(L) at position 463 to Asparagine (N); (b) substitution of Threonine (T)at position 508 to Arginine (R); (c) substitution of Isoleucine (I) atposition 523 to Aspartic Acid (D), Glutamic Acid (E), Glycine (G),Lysine (K), or Arginine (R); (d) substitution of Lysine (K) at position524 to Leucine (L); and, (e) a combination of two or more of saidsubstitutions, wherein said Tn3 scaffold has a plasma half-life longerthan the plasma half-life of a Tn3 scaffold not conjugated to said HSAvariant.

In some embodiments, the Tn3 scaffold comprises at least two identicalCD40L-specific monomer subunits. In other embodiments, the Tn3 scaffoldcomprises at least two different CD40L-specific monomer subunits. Insome embodiments, the Tn3 scaffold is a CD40 receptor agonist. In otherembodiments, the Tn3 scaffold is a CD40 receptor antagonist.

In some embodiments, the Tn3 scaffold comprises at least twoCD40L-specific monomer subunits which specifically bind to the sameCD40L epitope. In other embodiments, the Tn3 scaffold comprises at leasttwo CD40L-specific monomer subunits which specifically bind to differentCD40L epitopes. In some embodiments, these different CD40L epitopes arenon-overlapping epitopes. In other embodiments, these different CD40Lepitopes are overlapping epitopes.

In some embodiments, the Tn3 scaffold binds to at least two CD40Lmolecules. In other embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit which binds to at least two CD40Lmolecules.

In some embodiments, the beta strands of at least one CD40L-specificmonomer subunit of the Tn3 scaffold have at least 90% sequence identityto the beta strands of SEQ ID NO: 3. In other embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit comprising a A betastrand comprising SEQ ID NO: 11, or comprising a A beta strandcomprising SEQ ID NO: 11 except for at least one mutation. In otherembodiments, the Tn3 scaffold comprises a CD40L-specific monomer subunitcomprising a B beta strand comprising SEQ ID NO: 12, or comprising a Bbeta strand comprising SEQ ID NO: 12 except for at least one mutation.In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit comprising a C beta strand comprising SEQ ID NO: 13 or 14, orcomprising a C beta strand comprising SEQ ID NO: 13 or 14 except for atleast one mutation, and wherein the cysteine in SEQ ID NO: 13 or 14 isnot substituted. In other embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit comprising a D beta strand comprising SEQID NO: 15, or comprising a D beta strand comprising SEQ ID NO: 15 exceptfor at least one mutation.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit comprising an E beta strand comprising SEQ ID NO: 16, orcomprising an E beta strand comprising SEQ ID NO: 16 except for at leastone mutation. In other embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit comprising an F beta strand comprisingSEQ ID NO: 17, or comprising an F beta strand comprising SEQ ID NO: 17except for at least one mutation, and wherein the cysteine in SEQ ID NO:17 is not substituted. In some embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit comprising a G beta strand comprising SEQID NO: 18, or comprising a G beta strand comprising SEQ ID NO: 18 exceptfor at least one mutation.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit comprising the amino acid sequence:

(SEQ ID NO: 11) IEV (SEQ ID NO: 12) (X_(AB))_(n)ALITW (SEQ ID NO: 173)(X_(BC))_(n)CELX₁YGI (SEQ ID NO: 15) (X_(CD))nTTIDL (SEQ ID NO: 16)(X_(DE))_(n)YSI (SEQ ID NO: 17) (X_(EF))_(n)YEVSLIC (SEQ ID NO: 18)(X_(FG))_(n)KETFTT

-   -   wherein: X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG)        represent the amino acid residues present in the sequences of        the AB, BC, CD, DE, EF, and FG loops, respectively; X₁        represents amino acid residue A or T; and, the length of the        loop n is an integer between 2 and 26.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit, wherein the sequence of the AB loop comprises SEQ ID NO: 4 orSEQ ID NO: 136, the sequence of the CD loop comprises SEQ ID NO: 6, andthe sequence of the EF loop comprises SEQ ID NO: 8 or SEQ ID NO: 137. Inother embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit, wherein the sequence of the BC loop comprises a sequenceselected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, and 168. In some embodiments, the Tn3 scaffoldcomprises a CD40L-specific monomer subunit, wherein the sequence of theDE loop comprises a sequence selected from the group consisting of SEQID NOs: 94, 95, 96, 97, 98, and 169. In some embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit, wherein thesequence of the FG loop comprises a sequence selected from the groupconsisting of SEQ ID NOs: 9, 99, 139, and 170.

In other embodiments, the Tn3 scaffold comprises a CD40L-specificmonomer subunit, wherein the sequence of the BC loop comprises asequence selected from the group consisting of SEQ ID NOs: 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, and 174. In certain embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit, wherein the sequence of the DE loopcomprises a sequence selected from the group consisting of SEQ ID NOs:118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, and 175. In otherembodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit, wherein the sequence of the FG loop comprises a sequenceselected from the groups consisting of SEQ ID NOs: 129, 130, and 177. Incertain embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit, wherein the sequence of the AB loop comprises a sequenceselected from the group consisting of SEQ ID NOs: 4 and 136.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit, wherein the sequence of the BC loop comprises SEQ ID NO: 83,the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence ofthe FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the BC loop comprises SEQ ID NO: 83, the sequence of the DE loopcomprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQID NO: 99. In other embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit wherein the sequence of the BC loopcomprises SEQ ID NO: 84, the sequence of the DE loop comprises SEQ IDNO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 85, thesequence of the DE loop comprises SEQ ID NO: 94, and the sequence of theFG loop comprises SEQ ID NO: 9 or 139. In some embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the BC loop comprises SEQ ID NO: 86, the sequence of the DE loopcomprises SEQ ID NO: 96, and the sequence of the FG loop comprises SEQID NO: 9 or 139. In some embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit wherein the sequence of the BC loopcomprises SEQ ID NO: 87, the sequence of the DE loop comprises SEQ IDNO: 97, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 88, thesequence of the DE loop comprises SEQ ID NO: 95, and the sequence of theFG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the BC loop comprises SEQ ID NO: 89, the sequence of the DE loopcomprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQID NO: 9 or 139. In some embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit wherein the sequence of the BC loopcomprises SEQ ID NO: 90, the sequence of the DE loop comprises SEQ IDNO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139.

In other embodiments, the Tn3 scaffold comprises a CD40L-specificmonomer subunit wherein the sequence of the BC loop comprises SEQ ID NO:91, the sequence of the DE loop comprises SEQ ID NO: 95, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In someembodiments, the Tn3 scaffold comprises a CD40L-specific monomer subunitwherein the sequence of the BC loop comprises SEQ ID NO: 92, thesequence of the DE loop comprises SEQ ID NO: 98, and the sequence of theFG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the BC loop comprises SEQ ID NO: 93, the sequence of the DE loopcomprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQID NO: 9 or 139.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 100,the sequence of the DE loop comprises SEQ ID NO: 118, and the sequenceof the FG loop comprises SEQ ID NO: 129. In other embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the BC loop comprises SEQ ID NO: 101, the sequence of the DE loopcomprises SEQ ID NO: 119, and the sequence of the FG loop comprises SEQID NO: 129. In some embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit wherein the sequence of the BC loopcomprises SEQ ID NO: 102, the sequence of the DE loop comprises SEQ IDNO: 120, and the sequence of the FG loop comprises SEQ ID NO: 129. Inother embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 103,the sequence of the DE loop comprises SEQ ID NO: 121, and the sequenceof the FG loop comprises SEQ ID NO: 129. In some embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the AB loop comprises SEQ ID NO: 136.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 104,the sequence of the DE loop comprises SEQ ID NO: 122, and the sequenceof the FG loop comprises SEQ ID NO: 129. In other embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the BC loop comprises SEQ ID NO: 105, the sequence of the DE loopcomprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQID NO: 129. In other embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit wherein the sequence of the BC loopcomprises SEQ ID NO: 106, the sequence of the DE loop comprises SEQ IDNO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129. Insome embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the AB loop comprises SEQ ID NO: 136.

In certain embodiments, the Tn3 scaffold comprises a CD40L-specificmonomer subunit wherein the sequence of the BC loop comprises SEQ ID NO:107, the sequence of the DE loop comprises SEQ ID NO: 123, and thesequence of the FG loop comprises SEQ ID NO: 129. In other embodiments,the Tn3 scaffold comprises a CD40L-specific monomer subunit wherein thesequence of the BC loop comprises SEQ ID NO: 108, the sequence of the DEloop comprises SEQ ID NO: 118, and the sequence of the FG loop comprisesSEQ ID NO: 129. In some embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit wherein the sequence of the BC loopcomprises SEQ ID NO: 109, the sequence of the DE loop comprises SEQ IDNO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129. Insome embodiments, the sequence of the BC loop comprises SEQ ID NO: 110,the sequence of the DE loop comprises SEQ ID NO: 121, and the sequenceof the FG loop comprises SEQ ID NO: 129. In some embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the AB loop comprises SEQ ID NO: 136.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 111,the sequence of the DE loop comprises SEQ ID NO: 123, and the sequenceof the FG loop comprises SEQ ID NO: 130. In other embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the BC loop comprises SEQ ID NO: 108, the sequence of the DE loopcomprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQID NO: 129. In some embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit wherein the sequence of the BC loopcomprises SEQ ID NO: 112, the sequence of the DE loop comprises SEQ IDNO: 124, and the sequence of the FG loop comprises SEQ ID NO: 129. Insome embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 113,the sequence of the DE loop comprises SEQ ID NO: 125, and the sequenceof the FG loop comprises SEQ ID NO: 129. In some embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the AB loop comprises SEQ ID NO: 136.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 114,the sequence of the DE loop comprises SEQ ID NO: 118, and the sequenceof the FG loop comprises SEQ ID NO: 129. In other embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the BC loop comprises SEQ ID NO: 115, the sequence of the DE loopcomprises SEQ ID NO: 126, and the sequence of the FG loop comprises SEQID NO: 129. In some embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit wherein the sequence of the BC loopcomprises SEQ ID NO: 116, the sequence of the DE loop comprises SEQ IDNO: 127, and the sequence of the FG loop comprises SEQ ID NO: 129. Insome embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit wherein the sequence of the BC loop comprises SEQ ID NO: 117,the sequence of the DE loop comprises SEQ ID NO: 128, and the sequenceof the FG loop comprises SEQ ID NO: 129. In some embodiments, the Tn3scaffold comprises a CD40L-specific monomer subunit wherein the sequenceof the AB loop comprises SEQ ID NO: 136.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit comprising a sequence selected from the group consisting of SEQID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 146. In someembodiments, the Tn3 scaffold comprises a CD40L-specific monomer subunitcomprises the amino acid sequence:

(SEQ ID NO: 167) IEVKDVTDTTALITWX₁DX₂X₃X₄X₅X₆X₇X₈CELTYGIKDVPGDRTTIDLWX₉HX₁₀AX₁₁YSIGNLKPDTEYEVSLICRX₁₂GDMSSNPAKETFTT

-   -   wherein: (a) X₁ represents amino acid residue serine (S) or        leucine (L); (b) X₂ represents amino acid residue aspartic        acid (D) or glutamic acid (E); (c) X₃ represents amino acid        residue histidine (H), isoleucine (I), valine (V),        phenylalanine (F) or tryptophan (W); (d) X₄ represents amino        acid residue alanine (A), glycine (G), glutamic acid (E) or        aspartic acid (D); (e) X₅ represents amino acid residue glutamic        acid (E), leucine (L), glutamine (Q), serine (S), aspartic        acid (D) or asparagine (N); (f) X₆ represents amino acid residue        phenylalanine (F) or tyrosine (Y); (g) X₇ represents amino acid        residue isoleucine (I), valine (V), histidine (H), glutamic        acid (E) or aspartic acid (D); (h) X₈ represents amino acid        residue glycine (G), tryptophan (W) or valine (V); (i) X₉        represents amino acid residue tryptophan (W), phenylalanine (F)        or tyrosine (Y); (j) X₁₀ represents amino acid residue serine        (S), glutamine (Q), methionine (M) or histidine (H); (k) X₁₁        represents amino acid residue tryptophan (W) or histidine (H);        and, (l) X₁₂ represents amino acid residue arginine (R) or        serine (S).

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit comprising a sequence selected from the group consisting of SEQID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, and 82.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit comprising the amino acid sequence:

(SEQ ID NO: 171) IEVX₁DVTDTTALITWX₂X₃RSX₄X₅X₆X₇X₈X₉X₁₀CELX₁₁YGIKDVPGDRTTIDLX₁₂X₁₃X₁₄X₁₅YVHYSIGNLKPDTX₁₆YEVSLICLTTDGT YX₁₇NPAKETFTT 

-   -   wherein:    -   (a) X₁ represents amino acid residue lysine (K) or glutamic acid        (E);    -   (b) X₂ represents amino acid residue threonine (T) or isoleucine        (I);    -   (c) X₃ represents amino acid residue asparagine (N) or alanine        (A);    -   (d) X₄ represents amino acid residue serine (S), leucine (L),        alanine (A), phenylalanine (F) or tyrosine (Y);    -   (e) X₅ represents amino acid residue tyrosine (Y), alanine (A),        glycine (G), valine (V), isoleucine (I) or serine (S);    -   (f) X₆ represents amino acid residue tyrosine (Y), serine (S),        alanine (A) or histidine (H);    -   (g) X₇ represents amino acid residue asparagine (N), aspartic        acid (D), histidine (H) or tyrosine (Y);    -   (h) X₈ represents amino acid residue leucine (L), phenylalanine        (F), histidine (H) or tyrosine (Y);    -   (i) X₉ represents amino acid residue histidine (H), proline (P),        serine (S), leucine (L) or aspartic acid (D);    -   (j) X₁₀ represents amino acid residue glycine (G), phenylalanine        (F), histidine (H) or tyrosine (Y);    -   (k) X₁₁ represents amino acid residue alanine (A) or threonine        (T);    -   (l) X₁₂ represents amino acid residue serine (S), asparagine        (N), glutamic acid (E), asparagine (R) or aspartic acid (D);    -   (m) X₁₃ represents amino acid residue serine (S), glutamine (Q),        threonine (T), asparagine (N) or alanine (A);    -   (n) X₁₄ represents amino acid residue proline (P), valine (V),        isoleucine (I) or alanine (A) or no amino acid;    -   (o) X₁₅ represents amino acid residue isoleucine (I) or no amino        acid;    -   (p) X₁₆ represents amino acid residue glutamic acid (E) or        lysine (K); and,    -   (q) X₁₇ represents amino acid residue serine (S) or asparagine        (N).

In some embodiments, the Tn3 scaffold comprises a sequence selected fromthe group consisting of SEQ ID NOs: 134, 135, 205, 206, 207 and 208. Inother embodiments, the Tn3 scaffold consists of a sequence selected fromthe group consisting of SEQ ID NO: 134, 135, 205, 206, 207 and 208.

In some embodiments, the Tn3 scaffold comprises a sequence selected fromthe group consisting of SEQ ID NOs: 201, 202, 203, and 204. In someembodiments, the Tn3 scaffold consists of a sequence selected from thegroup consisting of SEQ ID NOs: 201, 202, 203, and 204. In someembodiments, the Tn3 scaffold comprises a CD40L-specific monomer subunitwherein said CD40L-specificity is towards human CD40L. In someembodiments, the Tn3 scaffold comprises a CD40L-specific monomer subunitwherein said CD40L-specificity is towards membrane bound CD40L (SEQ IDNO: 1), soluble CD40L (SEQ ID NO: 2), or a fragment thereof. In somespecific embodiments, the Tn3 scaffold binds CD40L and prevents bindingof CD40L to CD40.

The invention also provides a method of altering an activity in a CD40Lexpressing cell comprising contacting the cell with a Tn3 scaffold,wherein the Tn3 scaffold binds CD40L and prevents binding of CD40L toCD40. In some embodiments, the Tn3 scaffold binds to CD40L with anaffinity (K_(d)) of about 1 μM or less, or about 500 nM or less, orabout 100 nM or less, or about 50 nM or less, or about 25 nM or less, orabout 10 nM or less, or about 5 nM or less, or about 2 nM or less.

In some embodiments, the Tn3 scaffold comprises a CD40L-specific monomersubunit which specifically binds to a CD40L epitope comprising aminoacids located at positions 142 to 155, 200 to 230, or 247 to 251 of SEQID NO: 2. In some embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit that interacts with CD40L amino acidsE142, Y146, M148, N151, L155, R200, R203, and E230. In otherembodiments, the Tn3 scaffold comprises a CD40L-specific monomer subunitthat interacts with CD40L amino acids R203, I204, V247, H249, and T251.In other embodiments, the Tn3 scaffold comprises a CD40L-specificmonomer subunit that interacts with CD40L amino acids E142, Y146, M148,N151, L155, which are located in a first CD40L molecule, and with CD40Lamino acids R200, R203, and E230, which are located in a second CD40Lmolecule. In other embodiments, the Tn3 scaffold comprises aCD40L-specific monomer subunit that interacts with CD40L amino acidsR203 and I204, which are located in a first CD40L molecule, and withCD40L amino acids V247, H249, and T251, which are located in a secondCD40L molecule.

The invention also provides polypeptides comprising one or moreCD40L-specific Tn3 monomer, including but not limited to the serumalbumin fusions described herein.

The invention also provides an isolated nucleic acid molecule encoding aCD40L-specific Tn3 scaffold, an expression vector comprising the nucleicacid molecule encoding a CD40L-specific Tn3 scaffold, and a host cellcomprising such vector. The invention also provides a method ofproducing a Tn3 scaffold comprising culturing the host cell underconditions in which the CD40L-specific Tn3 scaffold encoded by thenucleic acid molecule is expressed.

The invention also provides a pharmaceutical composition comprising aCD40L-specific Tn3 scaffold and a pharmaceutically acceptable excipient.The invention also provides a method of preventing, treating,ameliorating, or managing autoimmune disease in a patient in needthereof comprising administering an effective amount of a pharmaceuticalcomposition comprising a CD40L-specific Tn3 scaffold.

The invention also provides a method of reducing the frequency orquantity of corticosteroid administered to a patient with an autoimmunedisease comprising administering to the patient a therapeuticallyeffective amount of a pharmaceutical composition comprising aCD40L-specific Tn3 scaffold.

The autoimmune disease treated by the administration of a CD40L-specificTn3 scaffold can be alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunediseases of the adrenal gland, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome,psoriasis, atherosclerosis, diabetic and other retinopathies,retrolental fibroplasia, age-related macular degeneration, neovascularglaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease),corneal and other tissue transplantation, and chronic inflammation,sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusioninjury, septicemia, endotoxic shock, cystic fibrosis, endocarditis,psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock,organ ischemia, reperfusion injury, spinal cord injury and allograftrejection. autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserythematosus, Meniere's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteritis/giant cell arteritis, ulcerative colitis,uveitis, vasculitis such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis.

In some specific embodiments, the autoimmune disease treated by theadministration of a CD40L-specific Tn3 scaffold is Systemic LupusErythematosus (SLE).

Methods of treatment with CD40L-specific Tn3 scaffolds can furthercomprise an additional therapy, such as immunotherapy, biologicaltherapy, chemotherapy, radiation therapy, or small molecule drugtherapy.

The invention also provides a protein crystal comprising a Tn3 scaffoldconsisting of SEQ ID NO: 20 in a complex with soluble CD40L (SEQ ID NO:2) wherein the crystal has a crystal lattice in a P2₁2₁2₁ orthorhombicspace group and unit cell dimensions, +/−0.1%, of a=85.69 Å, b=90.64 Å,c=95.56 Å. In some embodiments, the asymmetric unit of the crystalcomprises a trimer of CD40L and three molecules of Tn3 scaffold. Inother embodiments, the crystals diffract X-rays for a determination ofstructure coordinates to a resolution of a value equal to or less that3.2 Å.

The invention also provides a protein crystal comprising a Tn3 scaffoldconsisting of SEQ ID NO: 68 in a complex with soluble CD40L (SEQ ID NO:2) wherein the crystal has a crystal lattice in a P2₁3 cubic space groupand unit cell dimensions, +/−0.1%, of a=b=c=97.62 Å. In someembodiments, the asymmetric unit of the crystal comprises one CD40Lmolecule and one Tn3 scaffold molecule. In other embodiments, thecrystal diffracts X-rays for a determination of structure coordinates toa resolution of a value equal to or less than 2.7 Å.

The invention also provides a protein crystal comprising a Tn3 scaffoldconsisting of SEQ ID NO: 28 or 146 in a complex with soluble CD40L (SEQID NO: 2) wherein the crystal has a crystal lattice in a P321 spacegroup and unit cell dimensions, +/−0.1%, of a=95.53 Å, b=93.53 Å,c=66.69 Å. In some embodiments the asymmetric unit of the crystalcomprises one CD40L molecule and one Tn3 scaffold molecule. In otherembodiments the crystal diffracts X-rays for a determination ofstructure coordinates to a resolution of a value equal to or less than2.8 Å

The invention also provides a protein crystal comprising two differentTn3 scaffolds consisting of SEQ ID NO: 68 and SEQ ID NO: 28 or 146 in acomplex with soluble CD40L (SEQ ID NO: 2) wherein the crystal has acrystal lattice in a P2₁ cubic space group and unit cell dimensions,+/−0.1%, of a=80.32 Å, b=143.48 Å, c=111.27 Å, β=98.22 Å. In someembodiments the asymmetric unit of the crystal comprises two CD40Ltrimers and six of each Tn3 scaffold molecule. In other embodiments thecrystal diffracts X-rays for a determination of structure coordinates toa resolution of a value equal to or less than 1.9 Å

In some embodiments, the protein crystal is produced by usingsitting-drop vapor diffusion. The invention also provides a method ofmaking a protein crystal, comprising: (a) mixing a volume of a solutioncomprising a Tn3 scaffold comprising a CD40L-specific monomer subunit ina complex with CD40L with a volume of a reservoir solution comprising aprecipitant; and (b) incubating the mixture obtained in step (a) in aclosed container, under conditions suitable for crystallization untilthe protein crystal forms. In some embodiments, the method to producethe protein crystal comprises using sitting-drop vapor diffusion.

In some embodiments, the method to make a protein crystal is used toproduce crystals comprising the CD40L-specific Tn3 monomer subunits ofSEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 68 or SEQ ID NO: 146.

The invention also provides a machine-readable data storage mediumcomprising a data storage material encoded with machine-readableinstructions for: (a) transforming data into a graphicalthree-dimensional representation for the structure of a portion of aprotein crystal of a Tn3 scaffold comprising a CD40L-specific monomersubunit complexed with CD40L; and, (b) causing the display of saidgraphical three-dimensional representation. In some embodiments, suchTn3 scaffold comprises SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 68 orSEQ ID NO: 146. In other embodiments, such protein crystal is:

(a) a protein crystal comprising a Tn3 scaffold consisting of SEQ ID NO:20 in a complex with soluble CD40L (SEQ ID NO: 2) wherein the crystalhas a crystal lattice in a P2₁2₁2₁ orthorhombic space group and unitcell dimensions, +/−0.1%, of a=85.69 Å, b=90.64 Å, c=95.56 Å;

(b) a protein crystal comprising a Tn3 scaffold consisting of SEQ ID NO:68 in a complex with soluble CD40L (SEQ ID NO: 2) wherein the crystalhas a crystal lattice in a P2₁3 cubic space group and unit celldimensions, +/−0.1%, of a=b=c=97.62 Å; or

(c) a protein crystal comprising a Tn3 scaffold consisting of SEQ ID NO:20 and a Tn3 scaffold consisting of SEQ ID NO: 68, wherein both Tn3scaffold are in a complex with soluble CD40L (SEQ ID NO: 2)

(d) a protein crystal comprising a Tn3 scaffold consisting of SEQ ID NO:28 or 146 in a complex with soluble CD40L (SEQ ID NO: 2) wherein thecrystal has a crystal lattice in a P321 space group and unit celldimensions, +/−0.1%, of a=95.53 Å, b=93.53 Å, c=66.69 Å

(e) a protein crystal comprising two different Tn3 scaffolds consistingof SEQ ID NO: 68 and SEQ ID NO: 28 or 146 in a complex with solubleCD40L (SEQ ID NO: 2) wherein the crystal has a crystal lattice in a P2₁cubic space group and unit cell dimensions, +/−0.1%, of a=80.32 Å,b=143.48 Å, c=111.27 Å, β=98.22 Å.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1A shows the inhibition of murine CD40L (MuCD40L)-induced CD86expression measured using a D10G4.1/PBMC (Peripheral Blood mononuclearCell) assay. The M13 mouse CD40L-specific Tn3 scaffold, its M31 affinityoptimized variant (approximately 20× affinity improvement), theanti-CD40L MR1 monoclonal antibody, and a negative control were assayed.IC₅₀ values are also shown.

FIG. 1B shows CD40L inhibition in a murine NfkB assay. The assay usesNIHT3T cells expressing murine CD40R and containing an NfkB-Luciferasereporter construct. Addition of CD40L results in signaling (measured byluciferase activity) that is inhibited by both the MR1 anti-CD40Lantibody and by the M31 CD40L-specific Tn3 scaffold.

FIG. 2A shows the design of CD40L-specific tandem bivalent Tn3 scaffoldsand Serum Albumin (SA) fusion constructs.

FIG. 2B shows the SDS-PAGE analysis of a purified monovalent M13construct (CD40L-specific Tn3 construct), or tandem bivalent scaffoldswith linkers containing 1, 3, 5 or 7 Gly₄Ser units (denoted as GS)joining two M13 Tn3 monomer subunits. The monovalent M13 construct wasrun in lane 2, the dimeric construct with 1 GS unit (C1) was run inlanes 3 and 7, the dimeric constrict with 3 GS units (C2) was run inlanes 4 and 8, the dimeric construct with 5 GS units (C3) was run inlanes 5 and 9, and the dimeric construct with 7 GS units (C4) was run inlanes 6 and 10. Samples were run either non-reduced conditions (lanes2-6) or reduced conditions (lanes 7-10).

FIG. 2C shows the competitive inhibition of murine CD40L binding tomurine CD40 receptor immobilized on a biosensor chip by murineCD40L-specific monovalent (M13) or bivalent tandem scaffolds(M13-xGS-M13, wherein x is 1, 3, 5 or 7, corresponding to bivalentscaffolds with linkers containing 1, 3, 5 or 7 Gly₄Ser units). The halfmaximal inhibitory concentration (IC₅₀) for the various constructs isalso indicated.

FIG. 2D shows the inhibitory effect of murine CD40L-specific Tn3monovalent (M13) and bivalent tandem scaffolds, on murine CD40L-inducedCD86 expression on B cells. IC₅₀ values are provided for all Tn3constructs and for the MR1 anti-murine CD40L antibody.

FIG. 3A shows high expression levels of murine CD40L-specific tandembivalent Tn3 scaffold fused to mouse serum albumin (MSA) in HEK 293cells. These constructs have 1 (G₄S) repeat in the linker between theTn3 scaffold units and 3 (G₄S) repeats in the linker between the Tn3scaffold and MSA. In addition, the construct contains a N49Q mutationinto each of the M13 and M31 scaffolds to remove a potential N-linkedglycosylation site. 10 μl culture supernatant taken 3 or 6 days aftertransfection were run on an SDS-PAGE gel along with known quantities ofthe purified protein. Expression level was estimated to 200 mg/l 6 dayspost transfection. Purification was carried out by IMAC through aC-terminal His-tag.

FIG. 3B shows inhibition of murine CD40L (MuCD40L)-induced CD86expression measured using a D10G4.1/PBMC cell assay. A CD40L-specifictandem bivalent Tn3 scaffold (M13-1GS-M13), the same construct fused tomouse serum albumin (MSA) (M13-1GS-M13-MSA), and the MRI anti-murineCD40L monoclonal antibody were assayed. IC₅₀ values are provided for allconstructs.

FIG. 3C shows inhibition of murine CD40L (MuCD40L)-induced CD86expression measured using a D10G4.1/PBMC cell assay. A CD40L-specifictandem bivalent Tn3 scaffold (M13-1GS-M13) fused to mouse serum albumin(MSA) (M13-1GS-M13-MSA), an affinity matured variant of the M13 scaffoldconjugated to MSA (M31Mono-MSA), a tandem bivalent scaffold comprisingthe M31 affinity optimized variant conjugated to MSA (M31-1GS-M31-MSA),a negative control tandem bivalent scaffold that does not bind murineCD40L (D1-1GS-D1-MSA), and the MR1 monoclonal antibody were assayed.IC₅₀ values are provided.

FIG. 4A shows the pharmacokinetics of several murine CD40L specificconstructs in mouse as determined by ELISA. The plasma half-life(t_(1/2)) values for each construct are indicated.

FIG. 4B shows the pharmacokinetics of the human CD40L specific 342-HSAand a 342-HSA variant comprising the substitution of Leu at position 463with Asn (L463N) and the substitution of Lys at position 524 with Leu(K524L) in Cynomolgus monkey as determined by ELISA.

FIG. 5A shows B cell maturation in the germinal centers (GC) from asheep red blood cell (SRBC) immunization assay. The MR1 monoclonalanti-murine CD40L antibody was assayed.

FIG. 5B shows B cell maturation in the germinal centers (GC) from asheep red blood cell (SRBC) immunization assay. M31-derived monovalentand bivalent constructs fused to MSA were assayed. The D1-D1 bivalentconstruct conjugated to MSA was used a negative control.

FIG. 5C shows B cell maturation in periphery (nonGC) from a sheep redblood cell (SRBC) immunization assay. M31-derived monovalent andbivalent constructs fused to MSA were assayed. The D1-D1 bivalentconstruct conjugated to MSA was used a negative control.

FIG. 5D shows the percentage (% CD4) and number (#CD4) of CD4 positivecells from a sheep red blood cell (SRBC) immunization assay. M31-derivedmonovalent and bivalent constructs fused to MSA, and the MR1 anti-CD40Lmonoclonal antibodies were assayed. The D1-D1 bivalent constructconjugated to MSA was used a negative control.

FIG. 5E shows the percentage (% CD44hi) and number (#CD44hi) of CD44hipositive cells from a sheep red blood cell (SRBC) immunization assay.M31-derived monovalent and bivalent constructs fused to MSA and the MR1anti-CD40L monoclonal antibodies were assayed. The D1-D1 bivalentconstruct conjugated to MSA was used a negative control

FIG. 5F shows the amount of anti-SRBC IgG from a sheep red blood cell(SRBC) immunization assay. M31-derived monovalent and bivalentconstructs fused to MSA assayed. The D1-D1 bivalent construct conjugatedto MSA was used a negative control.

FIG. 5G shows the anti-KLH IgM titers from a KLH-specific T celldependent antibody response (TDAR) model. 342-derived monovalent andbivalent constructs fused to HSA were assayed.

FIG. 5H shows the anti-KLH IgG titers from a KLH-specific T celldependent antibody response (TDAR) model. 342-derived monovalent andbivalent constructs fused to HSA were assayed.

FIG. 6A shows the inhibitory effect of human CD40L-specific monovalentTn3 monomer scaffolds 309 and 311 on human CD40L-induced CD86 expressionon CD19 positive human PBMCs stimulated with Jurkat D1.1 cells.

FIG. 6B shows the inhibitory effect of human CD40L-specific monovalentTn3 monomer scaffolds 309 and 311 on human CD40L-stimulated B-cellproliferation.

FIG. 6C the inhibitory effect of human CD40L-specific monovalent Tn3monomer scaffolds 309 and 311 on plasma cell number in T/B cellco-cultures. Tn3 scaffold 309 was also shown to bind activated primary Tcells by FACS (data not shown). A D1 scaffold (“Neg Tn3”) was used ascontrol. Two monoclonal antibodies against CD40L, designated aCD40L(RE)and aCD40L(Bio) (Biogen's 5c8 anti-human CD40L monoclonal antibody) werealso used as controls.

FIG. 7A shows that human CD40L-specific monovalent Tn3 scaffolds 309 and311 have similar biophysical characteristics. Both scaffolds aremonodispersed as measured by SEC.

FIG. 7B shows that human CD40L-specific monovalent Tn3 scaffolds 309 and311 have similar biophysical characteristics. Both scaffolds havesimilar thermostability as the parent Tn3 scaffold (designated Tn3 (wildtype) in the graph) as measured by differential scanning calorimetry(DSC).

FIG. 8A shows inhibition of human CD40L-induced CD86 expression on CD19positive human PBMCs stimulated with Jurkat D1.1 cells. Monovalent (311)and bivalent (311_3GS and 311_7GS) human CD40L-specific Tn3 scaffoldswere assayed. IC₅₀ values for each construct are shown.

FIG. 8B shows inhibition of human CD40L-induced CD86 expression on CD19positive human PBMCs stimulated with Jurkat D1.1 cells. Monovalent (309)and bivalent (309_3GS and 309_7GS) human CD40L-specific Tn3 scaffoldswere assayed, as well as Biogen's 5c8 anti-human CD40L monoclonalantibody. IC₅₀ values for each construct and the antibody are shown.

FIG. 9A shows the design of a representative human CD40L-specific tandembivalent Tn3 scaffold fused to human serum albumin (HSA) (e.g., SEQ IDNO:135 or SEQ ID NO:145). “GGGGG” (SEQ ID NO: 148) and “GGGGA” (SEQ IDNO: 149) are alternative linkers to the “GGGGS” linkers (SEQ ID NO:147).

FIG. 9B shows a test purification from 293F cells over an IEX column.The shoulder fraction (<10% of the major peak) contains O-glycosylatedprotein linked to serine residues present in the linkers.

FIG. 9C shows inhibition in the human CD40L-induced CD86 expression onCD19 positive human PBMCs stimulated with Jurkat D1.1 cells. A bivalent(309) human CD40L-specific Tn3 scaffold, the same scaffold fused to HSA,and Biogen's 5c8 anti-human CD40L monoclonal antibody were assayed.

FIG. 9D shows inhibition in the human CD40L-induced CD86 expression onCD19 positive human PBMCs stimulated with Jurkat D1.1 cells. Threebivalent (309) human CD40L-specific Tn3 scaffolds were tested. Three(G₄S) repeats were present in the linker between the humanCD40L-specific subunits (309 in this example) while the linker betweenthe 309 subunits and the HSA was varied from 1 to 3 (G₄S) repeats.Biogen's 5c8 anti-human CD40L monoclonal antibody was also assayed.

FIG. 10A shows the effect of mutating loop sequences of 309 (left panel)and 311 (right panel) on CD40L binding. Binding indicates signalstrength in the binding assay. WT is the variant with the original leadsequence (parent Tn3 sequence), whereas BC, DE and FG denotes variantsin which the BC, DE, or FG loop sequence has been changed to the parentTn3 sequence as present in human Tenascin C.

FIG. 10B shows inhibition profiles of a panel of affinity optimizedscaffolds as measured by the human CD40L-induced CD86 expression on CD19positive human PBMCs stimulated with Jurkat D1.1 cells. HumanCD40L-specific Tn3 clone 309 monomers were affinity optimized. Affinityoptimized monomers are designated as clone 340 to clone 349. The clone309wtFG construct had the entire FG loop replaced with the FG loop ofthe parent Tn3 scaffold. The 5c8 anti-CD40L monoclonal antibody was alsoassayed.

FIG. 10C shows inhibition profiles as measured by the humanCD40L-induced CD86 expression on CD19 positive human PBMCs stimulatedwith Jurkat D1.1 cells. The profile of human CD40L-specific Tn3 311monomer, its K4E variant, and a negative control are shown.

FIG. 10D shows inhibition profiles as measured by the humanCD40L-induced CD86 expression on CD19 positive human PBMCs stimulatedwith Jurkat D1.1 cells. The profile of human CD40L-specific Tn3 311K4Emonomer, the affinity optimized 311K4E_12 monomer, and the 5c8anti-CD40L monoclonal antibody are shown. IC₅₀'s for the two constructsand the antibody are also presented.

FIG. 11A and FIG. 11B show a multiple sequence alignment of the parentalCD40L-specific Tn3 scaffold 309, the 309FGwt variant, and the affinityoptimized variants 340 to 349. Amino acid residues 1 to 42 are shown inFIG. 11A, and amino acid residues 43 to 83 are shown in FIG. 11B. Thevariant loops are shaded. The consensus amino acid sequence is presentedbelow the multiple sequence alignment. The aligned sequences correspondto the amino acid sequences of Tn3 scaffold clones 309 (SEQ ID NO: 20),309FGwt (SEQ ID NO: 22), 340 (SEQ ID NO: 24), 341 (SEQ ID NO: 26), 342(SEQ ID NO: 28), 343 (SEQ ID NO: 30), 344 (SEQ ID NO: 32), 345 (SEQ IDNO: 34), 346 (SEQ ID NO: 36), 347 (SEQ ID NO: 38), 348 (SEQ ID NO: 40),and 349 (SEQ ID NO: 42).

FIG. 12A and FIG. 12B show as multiple sequence alignment of theparental CD40L-specific Tn3 scaffold 311, the 311K4E variant, and theaffinity optimized variants 311K4E_1 to 311K4E_21. Amino acid residues 1to 44 are shown in FIG. 12A, and amino acid residues 45 to 87 are shownin FIG. 12B. The variant loops are shaded. Amino acid variations outsidethe shadowed loops are boxed. A consensus sequence is presented belowthe multiple sequence alignment. The aligned sequences correspond to theamino acid sequences of Tn3 scaffold clones 311 (SEQ ID NO: 44), 311K4E(SEQ ID NO: 46), 311K4E_1 (SEQ ID NO: 48), 311K4E_2 (SEQ ID NO: 50),311K4E_2 (SEQ ID NO: 52), 311K4E_3 (SEQ ID NO: 54), 311K4E_4 (SEQ ID NO:56), 311K4E_5 (SEQ ID NO: 58), 311K4E_7 (SEQ ID NO: 60), 311K4E_8 (SEQID NO: 62), 311K4E_9 (SEQ ID NO: 64), 311K4E_10 (SEQ ID NO: 66),311K4E_11 (SEQ ID NO: 68), 311K4E_12 (SEQ ID NO: 70), 311K4E_13 (SEQ IDNO: 72), 311K4E_14 (SEQ ID NO: 74), 311K4E_15 (SEQ ID NO: 76), 311K4E_16(SEQ ID NO: 78), 311K4E_19 (SEQ ID NO: 80), 311K4E_20 (SEQ ID NO: 82),and 311K4E_21 (SEQ ID NO: 84).

FIG. 13 shows a human NfkB inhibition assay that uses HEK293 cellsexpressing human CD40 receptor and containing an NfkB-Luciferasereporter construct. Addition of human CD40L results in signaling(measured by luciferase activity) that can be inhibited by CD40L bindingmolecules. The CD40L-specific Tn3 scaffolds 340 and 342, as well as the5c8 anti-CD40L monoclonal antibody were assayed.

FIG. 14 shows binding of human CD40L-specific Tn3 scaffolds to 24 hanti-CD3/28 activated human CD4+ T Cells. A monovalent 342 scaffoldfused to HSA (designated 342-HSA) and a bivalent 342 scaffold fused toHSA (designated 342-342-HSA) were assayed.

FIG. 15 shows inhibition of primary human T/B cell proliferation at day3. A monovalent 340 scaffold fused to HSA (340-HSA), a monovalent 342scaffold fused to HSA (342-HSA), and a bivalent 342 scaffold fused toHSA (342-342-HSA) were assayed. IC₅₀ values for each construct areshown.

FIG. 16A shows an aggregation assay on washed platelets. The graph showsa representative ADP induced aggregation positive control for a donor(Top three traces respectively) ADP: 0.5 μM, 1 μM, and 2 μM) along withthe immune complex (IC) of 5c8 monoclonal antibody (600 nM) and solublehuman CD40L (200 nM).

FIG. 16B shows an aggregation assay on washed platelets. The graph showslack of aggregation when preformed immuno complexes of 309-309 bivalentscaffolds (not fused to HSA) and soluble human CD40L were used. Theconcentration of human CD40L (soluble form) was kept constant at 600 nMand the concentration of the scaffold constructs was varied from 200 nMto 800 nM.

FIG. 16C an aggregation assay on washed platelets. The graph shows lackof aggregation when preformed immuno complexes of 342 monovalentscaffolds fused to HSA and soluble human CD40L were used. Theconcentration of human CD40L (soluble form) was kept constant at 600 nMand the concentration of the scaffold constructs was varied from 100 nMto 400 nM. The graph also shows rapid aggregation induced by the immunecomplex of Biogen 5c8 monoclonal antibody and soluble human CD40L.

FIG. 17A shows a ribbon representation of the crystal structure ofsoluble CD40L in a complex with the CD40L-specific Tn3 309 monomerscaffold. CD40L forms a trimer (polypeptides A, B and C). Each 309scaffold (polypeptides D, E and F, circled) makes contact with two CD40Lpolypeptides. The specific contacts between each 309 scaffold and thefirst and second CD40L polypeptides are listed. This is a “top-down”view of the structure.

FIG. 17B shows a ribbon representation of the crystal structure ofsoluble CD40L in a complex with the CD40L-specific Tn3 311K4E_12 monomerscaffold. CD40L forms a trimer (polypeptides A, B and C). Each 311K4E_12monomer scaffold (polypeptides D, E and F, circled) makes contact withtwo CD40L polypeptides. The specific contacts between each 311K4E_12monomer scaffold and the first and second CD40L polypeptides are listed.This is a “top-down” view of the structure.

FIG. 17C shows a ribbon representation illustrating that the 311K4E_12and 309 scaffolds (circled) bind to different epitopes located indifferent parts of the CD40L trimer complex. Both scaffolds bind in thesame groove that would interact with the CD40 receptor. This is a “side”view of the structure.

FIG. 17D shows a ribbon representation of the crystal structure ofsoluble CD40L in a complex with the CD40L-specific Tn3 342 monomerscaffold. Only one CD40L and one 342 monomer scaffold are shown. Thespecific contacts between the 342 monomer scaffold and the first CD40Lpolypeptides are listed.

FIG. 17E shows a ribbon representation illustrating that the 342 and311K4E_12 scaffolds can bind simultaneously to different epitopeslocated in different parts of the CD40L trimer complex. Both scaffoldsbind in the same groove that would interact with the CD40 receptor. Thisis a “side” view of the structure.

FIG. 18A shows the location of the contacts between amino acids in theCD40L-specific Tn3 311K4E_12 monomer scaffold (SEQ ID NO: 68) and atrimer formed by soluble CD40L (SEQ ID NO: 2) molecules as shown in FIG.17A. Each scaffold makes contact with 2 CD40L molecules. The CD40Lsequence (SEQ ID NO: 2) is shown. Dotted underline=cytoplasmic domain;Solid underline=signal anchor type II membrane protein; Doubleunderline=portion co-crystalized with Tn3 scaffold; Darkshading=residues on 1st CD40L that contact the Tn3; Lightshading=residues on 2nd CD40L that contact the Tn3.

FIG. 18B shows the location of the contacts between amino acids in theCD40L-specific Tn3 309 monomer scaffold and CD40L trimer as shown inFIG. 17B. Each scaffold makes contact with 2 CD40L molecules. The CD40Lsequence (SEQ ID NO: 2) is shown. Dotted underline=cytoplasmic domain;Solid underline=signal anchor type II membrane protein; Doubleunderline=portion co-crystalized with Tn3 scaffold; Darkshading=residues on 1st CD40L that contact the Tn3; Lightshading=residues on 2nd CD40L that contact the Tn3; double boxed residueis contact with FG loop of 309 scaffold, which likely is not conservedin clones having a wildtype FG loop.

FIG. 19 A. shows an exemplary chromatogram of the elution of Tn3scaffold (309 or 311K4E_12), CD40L and the complex between them off thesize exclusion Superdex 200 10/300 GL column. FIG. 19B shows crystals ofthe 309-CD40L complex. The crystal shown grew to dimensions up to0.15×0.15×0.1 mm. FIG. 19C shows crystals of the 311K4E_12-CD40Lcomplex.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such can vary. It must be noted that, as used inthis specification and the appended claims, the singular forms “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. The terms “a” (or “an”), as well as the terms “one or more,”and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A,”(alone) and “B” (alone). Likewise, the term “and/or” as used in a phrasesuch as “A, B, and/or C” is intended to encompass each of the followingembodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; Aand B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisinvention.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects orembodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

The term “epitope” as used herein refers to a protein determinantcapable of binding to a scaffold of the invention. Epitopes usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Conformational and non-conformational epitopes aredistinguished in that the binding to the former but not the latter islost in the presence of denaturing solvents.

The terms “fibronectin type III (FnIII) domain,” “FnIII domain” and“FnIII scaffold” refer to polypeptides homologous to the humanfibronectin type III domain having at least 7 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing solventexposed loops which connect the beta strands to each other. There are atleast three such loops at each edge of the beta sheet sandwich, wherethe edge is the boundary of the protein perpendicular to the directionof the beta strands. In certain embodiments, an FnIII domain comprises 7beta strands designated A, B, C, D, E, F, and G linked to six loopregions designated AB, BC, CD, DE, EF, and FG, wherein a loop regionconnects each beta strand.

The term “Tn3 scaffold” used herein, refers to molecules comprising atleast one FnIII scaffold wherein the A beta strand comprises SEQ ID NO:11, the B beta strand comprises SEQ ID NO: 12, the C beta strand SEQ IDNO: 13 or 14, the D beta strand comprises SEQ ID NO: 15, the E betastrand comprises SEQ ID NO: 16, the F beta strand comprises SEQ ID NO:17, and the beta strand G comprises SEQ ID NO: 18, wherein at least oneloop is a non-naturally occurring variant of the loops in the “parentTn3 scaffold.” In certain embodiments, one or more of the beta strandsof a Tn3 module comprise at least one amino acid substitution exceptthat the cysteine residues in the C beta strand (e.g., the cysteine inSEQ ID NOs: 13 or 14) and F beta strands (SEQ ID NO: 17) are notsubstituted.

The term “parent Tn3” as used herein refers to an FnIII scaffoldcomprising SEQ ID NO: 3, i.e., a thermally stabilizedcysteine-engineered FnIII scaffold derived from the 3rd FnIII domain ofhuman tenascin C.

The terms “multimer” or “multimeric scaffold” refer to a molecule thatcomprises at least two FnIII scaffolds in association. The scaffoldsforming a multimeric scaffold can be linked through a linker thatpermits each scaffold to function independently.

The terms “monomer,” “monomer subunit” or “monomer scaffold” refer to amolecule that comprises only one FnIII scaffold.

The term “CD40L-specific monomer subunit” as used herein refers to a Tn3monomer derived from a “parent Tn3” wherein the Tn3 monomer specificallybinds to CD40L or a fragment thereof, e.g., a soluble form of CD40L.

The term “DNA” refers to a sequence of two or more covalently bonded,naturally occurring or modified deoxyribonucleotides.

The term “fusion protein” refers to a protein that includes (i) one ormore scaffolds of the invention joined to (ii) a second, differentprotein (i.e., a “heterologous” protein).

TABLE 1  Sequences and SEQ ID NOs of components of “parent Tn3” SEQ IDName/Brief Description Sequence NO Tn3 IEVKDVTDTTALITWFKPLAEIDGCELT 3YGIKDVPGDRTTIDLTEDENQYSIGNLK PDTEYEVSLICRRGDMSSNPAKETFTT(cys residues of disulfide bond are underlined)3^(rd) FnIII of tenascin C, AB loop (Tn3) KDVTDTT 43^(rd) FnIII of tenascin C, BC loop (Tn3) FKPLAEIDG 53^(rd) FnIII of tenascin C, CD loop (Tn3) KDVPGDR 63^(rd) FnIII of tenascin C, DE loop (Tn3) TEDENQ 73^(rd) FnIII of tenascin C, EF loop (Tn3) GNLKPDTE 83^(rd) FnIII of tenascin C, FG loop (Tn3) RRGDMSSNPA 93^(rd) FnIII of tenascin C, beta strand A (Tn3) RLDASQIEV 103^(rd) FnIII of tenascin C, beta strand A (Tn3) IEV 11N-terminal truncation 3^(rd) FnIII of tenascin C, beta strand B (Tn3)ALITW 12 3^(rd) FnIII of tenascin C, beta strand C (Tn3  CELAYGI 13variant) 3^(rd) FnIII of tenascin C, beta strand C (Tn3) CELTYGI 143^(rd) FnIII of tenascin C, beta strand D (Tn3) TTIDL 153^(rd) FnIII of tenascin C, beta strand E (Tn3) YSI 163^(rd) FnIII of tenascin C, beta strand F (Tn3) YEVSLIC 173^(rd) FnIII of tenascin C, beta strand G (Tn3) KETFTT 18

The term “heterologous moiety” is used herein to indicate the additionof a composition to a Tn3 scaffold of the invention wherein thecomposition is not normally part of an FnIII domain. Exemplaryheterologous moieties include proteins, peptides, protein domains,linkers, drugs, toxins, imaging agents, radioactive compounds, organicand inorganic polymers, and any other compositions which might providean activity that is not inherent in the FnIII domain itself, including,but are not limited to, polyethylene glycol (PEG), a cytotoxic agent, aradionuclide, imaging agent, biotin, a dimerization domain (e.g. leucinezipper domain), human serum albumin (HSA) or an FcRn binding portionthereof, a domain or fragment of an antibody (e.g., antibody variabledomain, a CH1 domain, a Ckappa domain, a Clambda domain, a CH2, or a CH3domain), a single chain antibody, a domain antibody, an albumin bindingdomain, an IgG molecule, an enzyme, a ligand, a receptor, a bindingpeptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptidepolymer, a cytokine, and the like.

The term “linker” as used herein refers to any molecular assembly thatjoins or connects two or more scaffolds. The linker can be a moleculewhose function is to act as a “spacer” between modules in a scaffold, orit can also be a molecule with additional function (i.e., a “functionalmoiety”). A molecule included in the definition of “heterologous moiety”can also function as a linker.

The terms “linked” and “fused” are used interchangeably. These termsrefer to the joining together of two or more scaffolds, heterologousmoieties, or linkers by whatever means including chemical conjugation orrecombinant means.

The terms “domain” or “protein domain” refer to a region of a proteinthat can fold into a stable three-dimensional structure, oftenindependently of the rest of the protein, and which can be endowed witha particular function. This structure maintains a specific functionassociated with the domain's function within the original protein, e.g.,enzymatic activity, creation of a recognition motif for anothermolecule, or to provide necessary structural components for a protein toexist in a particular environment of proteins. Both within a proteinfamily and within related protein superfamilies, protein domains can beevolutionarily conserved regions. When describing the component of amultimeric scaffold, the terms “domain,” “monomeric scaffold,” “monomersubunit,” and “module” can be used interchangeably. By “native FnIIIdomain” is meant any non-recombinant FnIII domain that is encoded by aliving organism.

A “protein sequence” or “amino acid sequence” means a linearrepresentation of the amino acid constituents in a polypeptide in anamino-terminal to carboxyl-terminal direction in which residues thatneighbor each other in the representation are contiguous in the primarystructure of the polypeptide.

The term “nucleic acid” refers to any two or more covalently bondednucleotides or nucleotide analogs or derivatives. As used herein, thisterm includes, without limitation, DNA, RNA, and PNA. “Nucleic acid” and“polynucleotide” are used interchangeably herein.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). The term “isolated” nucleic acid or polynucleotide refers to anucleic acid molecule, DNA or RNA that has been removed from its nativeenvironment. For example, a recombinant polynucleotide encoding, e.g., ascaffold of the invention contained in a vector is considered isolatedfor the purposes of the present invention. Further examples of anisolated polynucleotide include recombinant polynucleotides maintainedin heterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, a polynucleotide or a nucleic acid can be or can include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

The term “pharmaceutically acceptable” refers to a compound or proteinthat can be administered to an animal (for example, a mammal) withoutsignificant adverse medical consequences.

The term “physiologically acceptable carrier” refers to a carrier whichdoes not have a significant detrimental impact on the treated host andwhich retains the therapeutic properties of the compound with which itis administered. One exemplary physiologically acceptable carrier isphysiological saline. Other physiologically acceptable carriers andtheir formulations are known to one skilled in the art and aredescribed, for example, in Remington's Pharmaceutical Sciences, (18thedition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.,incorporated herein by reference.

By a “polypeptide” is meant any sequence of two or more amino acidslinearly linked by amide bonds (peptide bonds) regardless of length,post-translation modification, or function. “Polypeptide,” “peptide,”and “protein” are used interchangeably herein. Thus, peptides,dipeptides, tripeptides, or oligopeptides are included within thedefinition of “polypeptide,” and the term “polypeptide” can be usedinstead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. A polypeptide can be generated in anymanner, including by chemical synthesis.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. Variants can occur naturally or be non-naturallyoccurring. Non-naturally occurring variants can be produced usingart-known mutagenesis techniques. Variant polypeptides can compriseconservative or non-conservative amino acid substitutions, deletions, oradditions. Also included as “derivatives” are those peptides thatcontain one or more naturally occurring amino acid derivatives of thetwenty standard amino acids.

By “randomized” or “mutated” is meant including one or more amino acidalterations, including deletion, substitution or addition, relative to atemplate sequence. By “randomizing” or “mutating” is meant the processof introducing, into a sequence, such an amino acid alteration.Randomization or mutation can be accomplished through intentional,blind, or spontaneous sequence variation, generally of a nucleic acidcoding sequence, and can occur by any technique, for example, PCR,error-prone PCR, or chemical DNA synthesis. The terms “randomizing”,“randomized”, “mutating”, “mutated” and the like are usedinterchangeably herein.

By a “cognate” or “cognate, non-mutated protein” is meant a protein thatis identical in sequence to a variant protein, except for the amino acidmutations introduced into the variant protein, wherein the variantprotein is randomized or mutated.

By “RNA” is meant a sequence of two or more covalently bonded, naturallyoccurring or modified ribonucleotides. One example of a modified RNAincluded within this term is phosphorothioate RNA.

The terms “scaffold of the invention” or “scaffolds of the invention” asused herein, refers to multimeric Tn3 scaffolds as well as monomeric Tn3scaffolds. The term “target” refers to a compound recognized by aspecific scaffold of the invention. The terms “target” and “antigen” areused interchangeably herein. The term “specificity” as used herein,e.g., in the terms “specifically binds” or “specific binding,” refers tothe relative affinity by which a Tn3 scaffold of the invention binds toone or more antigens via one or more antigen binding domains, and thatbinding entails some complementarity between one or more antigen bindingdomains and one or more antigens. According to this definition, a Tn3scaffold of the invention is said to “specifically bind” to an epitopewhen it binds to that epitope more readily than it would bind to arandom, unrelated epitope.

An “affinity matured” scaffold is a scaffold with one or morealterations, generally in a loop, which result in an improvement in theaffinity of the Tn3 scaffold for an epitope compared to a parent Tn3scaffold which does not possess those alteration(s).

The term “affinity” as used herein refers to a measure of the strengthof the binding of a certain Tn3 scaffold of the invention to anindividual epitope.

The term “avidity” as used herein refers to the overall stability of thecomplex between a population of Tn3 scaffolds of the invention and acertain epitope, i.e., the functionally combined strength of the bindingof a plurality of Tn3 scaffolds with the antigen. Avidity is related toboth the affinity of individual antigen-binding domains with specificepitopes, and also the valency of the scaffold of the invention.

The term “action on the target” refers to the binding of a Tn3 scaffoldof the invention to one or more targets and to the biological effectsresulting from such binding. In this respect, multiple antigen bindingunits in a Tn3 scaffold can interact with a variety of targets and/orepitopes and, for example, bring two targets physically closer, triggermetabolic cascades through the interaction with distinct targets, etc.With reference to CD40L, “action on the target” refers to the effectachieved, for example, by the enhancement, stimulation or activation, ofone or more biological activities of CD40L.

The term “valency” as used herein refers to the number of potentialantigen-binding modules, e.g., the number of FnIII modules in a scaffoldof the invention. When a Tn3 scaffold of the invention comprises morethan one antigen-binding module, each binding module can specificallybind, e.g., the same epitope or a different epitope, in the same targetor different targets.

The term “disulfide bond” as used herein includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup.

The term “immunoglobulin” and “antibody” comprises various broad classesof polypeptides that can be distinguished biochemically. Those skilledin the art will appreciate that heavy chains are classified as gamma,mu, alpha, delta, or epsilon. It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE,respectively. Modified versions of each of these classes are readilydiscernible to the skilled artisan. As used herein, the term “antibody”includes but not limited to an intact antibody, a modified antibody, anantibody VL or VL domain, a CH1 domain, a Ckappa domain, a Clambdadomain, an Fc domain (see below), a CH2, or a CH3 domain.

As used herein, the term “Fc domain” domain refers to a portion of anantibody constant region. Traditionally, the term Fc domain refers to aprotease (e.g., papain) cleavage product encompassing the paired CH2,CH3 and hinge regions of an antibody. In the context of this disclosure,the term Fc domain or Fc refers to any polypeptide (or nucleic acidencoding such a polypeptide), regardless of the means of production,that includes all or a portion of the CH2, CH3 and hinge regions of animmunoglobulin polypeptide.

As used herein, the term “modified antibody” includes synthetic forms ofantibodies which are altered such that they are not naturally occurring,e.g., antibodies that comprise at least two heavy chain portions but nottwo complete heavy chains (as, e.g., domain deleted antibodies orminibodies); multispecific forms of antibodies (e.g., bispecific,trispecific, etc.) altered to bind to two or more antigens or todifferent epitopes of a single antigen). In addition, the term “modifiedantibody” includes multivalent forms of antibodies (e.g., trivalent,tetravalent, etc., antibodies that to three or more copies of the sameantigen). (See, e.g., Antibody Engineering, Kontermann & Dubel, eds.,2010, Springer Protocols, Springer).

The term “in vivo half-life” is used in its normal meaning, i.e., thetime at which 50% of the biological activity of a polypeptide is stillpresent in the body/target organ, or the time at which the activity ofthe polypeptide is 50% of its initial value. As an alternative todetermining functional in vivo half-life, “serum half-life” may bedetermined, i.e., the time at which 50% of the polypeptide moleculescirculate in the plasma or bloodstream prior to being cleared.Determination of serum-half-life is often more simple than determiningfunctional in vivo half-life and the magnitude of serum-half-life isusually a good indication of the magnitude of functional in vivohalf-life. Alternative terms to serum half-life include “plasmahalf-life,” circulating half-life, circulatory half-life, serumclearance, plasma clearance, and clearance half-life. The functionalityto be retained is normally selected from procoagulant, proteolytic,co-factor binding, receptor binding activity, or other type ofbiological activity associated with the particular protein.

The term “increased” with respect to the functional in vivo half-life orplasma half-life is used to indicate that the relevant half-life of thepolypeptide is statistically significantly increased relative to that ofa reference molecule (for example an unmodified polypeptide), asdetermined under comparable conditions.

The term “decreased” with respect to the functional in vivo half-life orplasma half-life is used to indicate that the relevant half-life of thepolypeptide is statistically significantly decreased relative to that ofa reference molecule (for example an unmodified polypeptide), asdetermined under comparable conditions.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a scaffold of the invention or afragment thereof. The process includes any manifestation of thefunctional presence of the gene within the cell including, withoutlimitation, gene knockdown as well as both transient expression andstable expression. It includes without limitation transcription of thegene into one or more mRNAs, and the translation of such mRNAs into oneor more polypeptides. If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.

An “expression product” can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide. Expressionproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation, or polypeptideswith post translational modifications, e.g., methylation, glycosylation,the addition of lipids, association with other protein subunits,proteolytic cleavage, and the like.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired expression product in a hostcell. As known to those skilled in the art, such vectors can easily beselected from the group consisting of plasmids, phages, viruses andretroviruses. In general, vectors compatible with the instant inventionwill comprise a selection marker, appropriate restriction sites tofacilitate cloning of the desired nucleic acid and the ability to enterand/or replicate in eukaryotic or prokaryotic cells.

The term “host cells” refers to cells that harbor vectors constructedusing recombinant DNA techniques and encoding at least one expressionproduct. In descriptions of processes for the isolation of an expressionproduct from recombinant hosts, the terms “cell” and “cell culture” areused interchangeably to denote the source of the expression productunless it is clearly specified otherwise, i.e., recovery of theexpression product from the “cells” means either recovery from spun downwhole cells, or recovery from the cell culture containing both themedium and the suspended cells.

The terms “treat” or “treatment” as used herein refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder in a subject, such as the progressionof an inflammatory disease or condition. Beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable.

The term “treatment” also means prolonging survival as compared toexpected survival if not receiving treatment. Those in need of treatmentinclude those already with the condition or disorder as well as thoseprone to have the condition or disorder or those in which the conditionor disorder is to be prevented.

The terms “subject,” “individual,” “animal,” “patient,” or “mammal”refer to any individual, patient or animal, in particularly a mammaliansubject, for whom diagnosis, prognosis, or therapy is desired. Mammaliansubjects include humans, domestic animals, farm animals, and zoo,sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats,mice, horses, cattle, cows, and so on.

The term “CD40L” as used herein refers without limitations to CD40Lexpressed on the surface of T-cells, recombinantly expressed CD40L,CD40L expressed and purified form E. coli or other suitable recombinantprotein expression systems, aglycosylated CD40L, and soluble fragmentsof CD40L. As used herein, “CD40L” also refers to MegaCD40L. MegaCD40L™is a high activity construct in which two trimeric CD40 ligands areartificially linked via the collagen domain of ACRP30/adiponectin. Thisconstruct very effectively simulates the natural membrane-assistedaggregation of CD40L in vivo. It provides a simple and equally potentalternative to [CD40L+enhancer] combinations (Alexis biochemicals). Theterm “CD40L” refers to monomeric forms of CD40L as well as oligomericforms, e.g., trimeric CD40L.

The term “CD40L” refers both to the full length CD40L and to solublefragments, e.g., extracellular domain forms of CD40L resulting fromproteolysis. Amino acid sequences of membrane-bound and soluble forms ofhuman CD40L (Swissprot: P29965) are shown in SEQ ID NO: 1 and SEQ ID NO:2, respectively.

The terms “CD40L antagonist” or “antagonist” are used in the broadestsense, and includes any molecule that partially or fully inhibits,decreases or inactivates one or more biological activities of CD40L, andbiologically active variants thereof, in vitro, in situ, or in vivo. Forinstance, a CD40L antagonist may function to partially or fully inhibit,decrease or inactivate one or more biological activities of one or moreCD40L molecules, or one or more CD40L molecules bound to CD40 or othertargets, in vivo, in vitro or in situ, as a result of its binding toCD40L.

The term “CD40L agonist” or “agonist” is used in the broadest sense, andincludes any molecule that partially or fully enhances, stimulates oractivates one or more biological activities of CD40L, and biologicallyactive variants thereof, in vitro, in situ, or in vivo. For instance, aCD40L agonist may function to partially or fully enhance, stimulate oractivate one or more biological activities of one or more CD40Lmolecules, or one or more CD40L molecules bound to CD40R or othertargets, in vivo, in vitro or in situ, as a result of its binding toCD40L.

The term “crystal” as used herein, refers to one form of solid state ofmatter in which atoms are arranged in a pattern that repeatsperiodically in three-dimensions, typically forming a lattice.

The term “space group symmetry,” as used herein, refers to the wholesymmetry of the crystal that combines the translational symmetry of acrystalline lattice with the point group symmetry. A “space group” isdesignated by a capital letter identifying the lattice group (P, A, F,etc.) followed by the point group symbol in which the rotation andreflection elements are extended to include screw axes and glide planes.Note that the point group symmetry for a given space group can bedetermined by removing the cell centering symbol of the space group andreplacing all screw axes by similar rotation axes and replacing allglide planes with mirror planes. The point group symmetry for a spacegroup describes the true symmetry of its reciprocal lattice.

The term “unit cell,” as used herein, means the atoms in a crystal thatare arranged in a regular repeated pattern, in which the smallestrepeating unit is called the unit cell. The entire structure can bereconstructed from knowledge of the unit cell, which is characterized bythree lengths (a, b, and c) and three angles (α, β, and γ). Thequantities a and b are the lengths of the sides of the base of the celland γ is the angle between these two sides. The quantity c is the heightof the unit cell. The angles α and β describe the angles between thebase and the vertical sides of the unit cell.

The term “machine-readable data storage medium,” as used herein, means adata storage material encoded with machine-readable data, wherein amachine is programmed with instructions for using such data and iscapable of displaying data in the desired format, for example, agraphical three-dimensional representation of molecules or molecularcomplexes.

The term “X-ray diffraction pattern” means the pattern obtained fromX-ray scattering of the periodic assembly of molecules or atoms in acrystal. X-ray crystallography is a technique that exploits the factthat X-rays are diffracted by crystals. X-rays have the properwavelength (in the Angstrom range, approximately 10⁻⁸ cm) to bescattered by the electron cloud of an atom of comparable size. Based onthe diffraction pattern obtained from X-ray scattering of the periodicassembly of molecules or atoms in the crystal, the electron density canbe reconstructed. Additional phase information can be extracted eitherfrom the diffraction data or from supplementing diffraction experimentsto complete the reconstruction (the phase problem in crystallography). Amodel is the progressively built into the experimental electron density,refined against the data to produce an accurate molecular structure.X-ray structure coordinates define a unique configuration of points inspace. Those of skill in the art understand that a set of structurecoordinates for a protein or a protein-ligand complex, or a portionthereof, define a relative set of points that, in turn, define aconfiguration in three dimensions. A similar or identical configurationcan be defined by an entirely different set of coordinates, provided thedistances and angles between coordinates remain essentially the same. Inaddition, a configuration of points can be defined by increasing ordecreasing the distances between coordinates by a scalar factor, whilekeeping the angles essentially the same.

The term “crystal structure,” as used herein, refers to thethree-dimensional or lattice spacing arrangement of repeating atomic ormolecular units in a crystalline material. The crystal structure of acrystalline material can be determined by X-ray crystallographicmethods, see, for example, “Principles of Protein X-Ray Crystallography”by Jan Drenth, Springer Advanced Texts in Chemistry, Springer Verlag,2nd ed., February 199, ISBN: 0387985875, and “Introduction toMacromolecular Crystallography” by Alexander McPherson, Wiley-Liss, Oct.18, 2002, ISBN: 0471251224.

The term “effector function” refers to those biological activities of anantibody or antibody fragment attributable to the Fc region (a native Fcregion or amino acid sequence variant Fc region) of an antibody, andvary with the antibody isotype. Examples of antibody effector functionsinclude: C1q binding and complement dependent cytotoxicity; Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; downregulation of cell surface receptors (e.g., B cellreceptors); and B cell activation.

The term “antibody-dependent cell-mediate cytotoxicity” or “ADCC” refersto a form of cytotoxicity in which secreted Ig bound onto Fc receptors(FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK)cells, neutrophils, and macrophages) enable these cytotoxic effectorcells to bind specifically to an antigen-bearing target cell andsubsequently kill the target cells with cytotoxins.

The term “Fe receptor” or “FcR” describes a receptor that binds to theFc region of an antibody. The FcR can be a native sequence human FcR.The FcR can bind to an IgG antibody (a gamma receptor) and includesreceptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelicvariants and alternatively spliced forms of these receptors. The termalso includes the neonatal receptor FcRn.

The term “consensus sequence” refers to a protein sequence showing themost common amino acids at a particular position after multiplesequences are aligned. A consensus sequence is a way of representing theresults of a multiple sequence alignment, where related sequences arecompared to each other. The consensus sequence shows which residues aremost abundant in the alignment at each position, and the degree ofvariability at each position.

Introduction

CD40L (also known as CD154, CD40 ligand, gp39 or TBAM) is a 33 kDa, TypeII membrane glycoprotein (Swiss-ProtAce-No P29965). Additionally,shorter 18 kDa CD40L soluble forms exist, (also known as sCD40L orsoluble CD40L). These soluble forms of CD40L are generated byproteolytic processing of the membrane bound protein, but the cellularactivity of the soluble species is weak in the absence of higher orderoligomerization (e.g., trimerization).

The present invention provides a family of recombinant, non-naturallyoccurring protein scaffolds (Tn3 scaffolds) capable of binding to CD40L.In particular, the proteins described herein can be used to displaydefined loops which are analogous to the complementarity-determiningregions (“CDRs”) of an antibody variable region. These loops can besubjected to randomization or restricted evolution to generate diversitycapable of binding to a multitude of target compounds. The Tn3 scaffoldscan be used as monomers or can be assembled into multimer scaffoldscapable of binding to CD40L.

In specific embodiments, the invention provides CD40L-specific binderswhich are useful for preventing ameliorating, detecting, diagnosing, ormonitoring diseases, such as but not limited to autoimmune disease. Inother specific embodiments, CD40L-specific Tn3 scaffolds of theinvention are useful for the treatment of autoimmune diseases andconditions. In some embodiments, autoimmune diseases may include, butare not limited to systemic lupus erythematosus (SLE), rheumatoidarthritis (RA), multiple sclerosis (MS), inflammatory bowel disease(IBD) and allograft rejection.

The Tn3 scaffolds of the invention comprise CD40L-specific monomersubunits derived from the third FnIII domain of human tenascin C, inwhich at least one non-naturally occurring intramolecular disulfide bondhas been engineered. The monomer subunits that make up the Tn3 scaffoldsof the invention correctly fold independently of each other, retaintheir binding specificity and affinity, and each of the monomericscaffolds retains its functional properties. When monomer subunits areassembled in high valency multimeric Tn3 scaffolds the monomer subunitscorrectly fold independently of each other, retain their bindingspecificity and affinity, and each one of the monomers retains itsfunctional properties.

Tn3 scaffolds of the invention comprising more than one monomer subunitcan bind to multiple epitopes, e.g., (i) bind to multiple epitopes in asingle target, (ii) bind to a single epitope in multiple targets, (iii)bind to multiple epitopes located on different subunits of one target,or (iv) bind to multiple epitopes on multiple targets, thus increasingavidity.

In addition, due to the possibility of varying the distance betweenmultiple monomers via linkers, multimeric Tn3 scaffolds are capable ofbinding to multiple target molecules on a surface (either on the samecell/surface or in different cells/surfaces). As a result of theirability to bind simultaneously to more than one target, a Tn3 multimericscaffold of the invention can be used to modulate multiple pathways,cross-link receptors on a cell surface, bind cell surface receptors onseparate cells, and/or bind target molecules or cells to a substrate.

In addition, the present invention provides affinity matured scaffoldswherein the affinity of a scaffold for a specific target is modulatedvia mutation. Also, the invention provides methods to produce thescaffolds of the invention as well as methods to engineer scaffolds withdesirable physicochemical, pharmacological, or immunological properties.Furthermore, the present invention provides uses for such scaffolds andmethods for therapeutic, prophylactic, and diagnostic use.

The FnIII Structural Motif

The Tn3 scaffolds of the present invention are based on the structure ofa type III fibronectin module (FnIII), a domain found widely across allthree domains of life and viruses, and in multitude of protein classes.In specific embodiments, the scaffolds of the invention are derived fromthe third FnIII domain of human tenascin C (see InternationalApplication No. International Application No. PCT/US2008/012398,published as WO 2009/058379; PCT/US2011/032184, published as WO2011/130324; and International Application No. PCT/US2011/032188,published as WO2011130328).

In one specific embodiment, the Tn3 scaffolds of the invention comprisea CD40L-specific monomer subunit derived from a parent Tn3 scaffold. Theoverall tridimensional fold of the monomer is closely related to that ofthe smallest functional antibody fragment, the variable region of theheavy chain (VH), which in the single domain antibodies of camels andcamelids (e.g., llamas) comprises the entire antigen recognition unit.

The Tn3 monomer subunits of the invention and the native FnIII domainfrom tenascin C are characterized by the same tridimensional structure,namely a beta-sandwich structure with three beta strands (A, B, and E)on one side and four beta strands (C, D, F, and G) on the other side,connected by six loop regions. These loop regions are designatedaccording to the beta-strands connected to the N- and C-terminus of eachloop. Accordingly, the AB loop is located between beta strands A and B,the BC loop is located between strands B and C, the CD loop is locatedbetween beta strands C and D, the DE loop is located between betastrands D and E, the EF loop is located between beta strands E and F,and the FG loop is located between beta strands F and G. FnIII domainspossess solvent exposed loop s tolerant of randomization, whichfacilitates the generation of diverse pools of protein scaffolds capableof binding specific targets with high affinity.

In one aspect of the invention, Tn3 monomer subunits are subjected todirected evolution designed to randomize one or more of the loops whichare analogous to the complementarity-determining regions (CDRs) of anantibody variable region. Such a directed evolution approach results inthe production of antibody-like molecules with high affinities fortargets of interest, e.g., CD40L.

In addition, in some embodiments the Tn3 scaffolds described herein canbe used to display defined exposed loops (for example, loops previouslyrandomized and selected on the basis of target binding) in order todirect the evolution of molecules that bind to such introduced loops.This type of selection can be carried out to identify recognitionmolecules for any individual CDR-like loop or, alternatively, for therecognition of two or all three CDR-like loops combined into a nonlinearepitope binding moiety. A set of three loops (designated BC, DE, andFG), which can confer specific target binding, run between the B and Cstrands; the D and E strands, and the F and G beta strands,respectively. The BC, DE, and FG loops of the third FnIII domain ofhuman tenascin C are 9, 6, and 10 amino acid residues long,respectively. The length of these loops falls within the narrow range ofthe cognate antigen-recognition loops found in antibody heavy chains,that is, 7-10, 4-8, and 4-28 amino acids in length, respectively.Similarly, a second set of loops, the AB, CD, and EF loops (7, 7, and 8,amino acids in length respectively) run between the A and B betastrands; the C and D beta strands; and the E and F beta strands,respectively.

Once randomized and selected for high affinity binding to a target, theloops in the Tn3 monomer scaffold may make contacts with targetsequivalent to the contacts of the cognate CDR loops in antibodies.Accordingly, in some embodiments the AB, CD, and EF loops are randomizedand selected for high affinity binding to one or more targets, e.g.,CD40L. In some embodiments, this randomization and selection process maybe performed in parallel with the randomization of the BC, DE, and FGloops, whereas in other embodiments this randomization and selectionprocess is performed in series.

CD40L-Specific Monomeric Subunits

The invention provides CD40L-specific recombinant, non-naturallyoccurring Tn3 scaffolds comprising, a plurality of beta strand domainslinked to a plurality of loop regions, wherein one or more of said loopregions vary by deletion, substitution or addition of at least one aminoacid from the cognate loops in wild type Tn3 (SEQ ID NO: 3) (see TABLE1).

To generate improved CD40L-specific Tn3 monomer subunits with novelbinding characteristics, parent Tn3 is subjected to amino acidadditions, deletions or substitutions. It will be understood that, whencomparing the sequence of a CD40L-specific Tn3 monomer subunit to thesequence of parent Tn3, the same definition of the beta strands andloops is utilized. In some embodiments, the CD40L-specific Tn3 monomersubunits of the invention comprise the amino acid sequence:

(SEQ ID NO: 11) IEV (SEQ ID NO: 12) (X_(AB))_(n)ALITW (SEQ ID NO: 173)(X_(BC))_(n)CELX₁YGI (SEQ ID NO: 15) (X_(CD))_(n)TTIDL (SEQ ID NO: 16)(X_(DE))_(n)YSI (SEQ ID NO: 17) (X_(EF))_(n)YEVSLIC (SEQ ID NO: 18)(X_(FG))_(n)KETFTT

-   -   wherein:    -   (a) X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent        the amino acid residues present in the sequences of the AB, BC,        CD, DE, EF, and FG loops, respectively;    -   (b) X₁ represents amino acid residue alanine (A) or threonine        (T); and,    -   (c) length of the loop n is an integer between 2 and 26.

TABLE 2 Loop Sequences of Tn3 Clones Used in These Studies AB Loop BCLoop CD Loop DE Loop EF Loop FG Loop Clone SEQ ID NO SEQ ID NO SEQ ID NOSEQ ID NO SEQ ID NO SEQ ID NO* PARENT Tn3 Tn3  4  5 6  7  8  9 309FAMILY 309FGwt  4  83 6  94  8  9 309  4  83 6  94  8  99 340  4  84 6 95  8  9 341  4  85 6  94  8  9 342  4  86 6  96  8  9 343  4  87 6  97 8  9 344  4  88 6  95  8  9 345  4  89 6  94  8  9 346  4  90 6  94  8 9 347  4  91 6  95  8  9 348  4  92 6  98  8  9 349  4  93 6  94  8  9309FGwt  4 168 6 169  8 170 consensus 311 FAMILY** 311  4 100 6 118  8129 311K4E 136 100 6 118 137 129 311K4E_1 136 101 6 119  8 129 311K4E_2136 102 6 120  8 129 311K4E_3† 136 103 6 121  8 129 311K4E_4† 136 104 6122  8 129 311K4E_5† 136 105 6 121  8 129 311K4E_7 136 106 6 123  8 129311K4E_8† 136 107 6 123  8 129 311K4E_9 136 108 6 118  8 129 311K4E_10†136 109 6 123  8 129 311K4E_11 136 110 6 121  8 129 311K4E_12† 136 111 6123  8 130 311K4E_13 136 108 6 121  8 129 311K4E_14 136 112 6 124  8 129311K4E_15 136 113 6 125  8 129 311K4E_16 136 114 6 118  8 129 311K4E_19136 115 6 126  8 129 311K4E_20 136 116 6 127  8 129 311K4E_21 136 117 6128  8 129 311 consensus 173 174 6 175 176 177 †Clones comprising a Cbeta strand having the sequence CELAYGI (SEQ ID NO: 14), all otherclones comprising a C beta strand having the sequence CELTYGI (SEQ IDNO: 13). *In some variants in the 309 family, e.g., 342, the FG loop canbe replaced with SEQ ID NO: 139. **In some variants in the 311 family,the BC loop can be engineered to replace the tyrosine at position 21. Itis specifically contemplated that the replacement amino acid residuescan have a small side chain.

In some embodiments, the CD40L-specific Tn3 monomer subunits of theinvention consist of the amino acid sequence:

(SEQ ID NO: 11) IEV (SEQ ID NO: 12) (X_(AB))_(n)ALITW (SEQ ID NO: 173)(X_(BC))_(n)CELX₁YGI (SEQ ID NO: 15) (X_(CD))_(n)TTIDL (SEQ ID NO: 16)(X_(DE))_(n)YSI (SEQ ID NO: 17) (X_(EF))_(n)YEVSLIC (SEQ ID NO: 18)(X_(FG))_(n)KETFTT

-   -   wherein:    -   (a) X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent        the amino acid residues present in the sequences of the AB, BC,        CD, DE, EF, and FG loops, respectively;    -   (b) X₁ represents amino acid residue alanine (A) or threonine        (T); and,    -   (c) length of the loop n is an integer between 2 and 26.

In one embodiment, the beta strands of the CD40L-specific Tn3 monomerscaffold have at least 90% sequence identity to the beta strands of theparent Tn3 scaffold (SEQ ID NO: 3). To calculate such percentage ofsequence identify, amino acid sequences are aligned applying methodsknown in the art. The percentage of sequence identity is defined as theratio between (a) the number of amino acids located in beta strandswhich are identical in the sequence alignment and (b) the total numberof amino acids located in beta strands.

In one embodiment, the sequence of the AB loop comprises SEQ ID NO: 4 orSEQ ID NO: 136. In another embodiment, the sequence of the CD loopcomprises SEQ ID NO: 6. In another embodiment, the sequence of the EFloop comprises SEQ ID NO: 8 or SEQ ID NO: 137. In one embodiment, thesequence of the AB loop consists of SEQ ID NO: 4 or SEQ ID NO: 136. Inanother embodiment, the sequence of the CD loop consists of SEQ ID NO:6. In another embodiment, the sequence of the EF loop consists of SEQ IDNO: 8 or SEQ ID NO: 137.

In one embodiment, the sequence of the BC loop comprises a sequenceselected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87,88, 89, 90, 91, 92 and 93. In another embodiment, the sequence of the BCloop consists of a sequence selected from the group consisting of SEQ IDNOs: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 and 93.

In one embodiment, the sequence of the DE loop comprises a sequenceselected from the group consisting of SEQ ID NOs: 94, 95, 96, 97 and 98.In another embodiment, the sequence of the DE loop consists of asequence selected from the group consisting of SEQ ID NOs: 94, 95, 96,97 and 98.

In one embodiment, the sequence of the FG loop comprises a sequenceselected from the group consisting of SEQ ID NOs: 9, 99, and 139. Inanother embodiment, the sequence of the FG loop consists of a sequenceselected from the group consisting of SEQ ID NOs: 9, 99, and 139.

In one embodiment, the sequence of the BC loop comprises a sequenceselected from the group consisting of SEQ ID NOs: 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 and 117.In another embodiment, the sequence of the BC loop consists of asequence selected from the group consisting of SEQ ID NOs: 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116 and 117.

In some embodiments, the sequence of the DE loop comprises a sequenceselected from the group consisting of SEQ ID NOs: 118, 119, 120, 121,122, 123, 124, 125, 126, 127 and 128. In other embodiments, the sequenceof the DE loop consists of a sequence selected from the group consistingof SEQ ID NOs: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 and 128.

In some embodiments, the sequence of the FG loop comprises a sequenceselected from the groups consisting of SEQ ID NOs: 129 and 130. In otherembodiments, the sequence of the FG loop consists of a sequence selectedfrom the groups consisting of SEQ ID NOs: 129 and 130.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:83, the sequence of the DE loop comprises SEQ ID NO: 94, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In someembodiments, the sequence of the BC loop consists of SEQ ID NO: 83, thesequence of the DE loop consists of SEQ ID NO: 94, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:83, the sequence of the DE loop comprises SEQ ID NO: 94, and thesequence of the FG loop comprises SEQ ID NO: 99. In other embodiments,the sequence of the BC loop consists of SEQ ID NO: 83, the sequence ofthe DE loop consists of SEQ ID NO: 94, and the sequence of the FG loopconsists of SEQ ID NO: 99.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:84, the sequence of the DE loop comprises SEQ ID NO: 95, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 84, thesequence of the DE loop consists of SEQ ID NO: 95, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:85, the sequence of the DE loop comprises SEQ ID NO: 94, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 85, thesequence of the DE loop consists of SEQ ID NO: 94, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:86, the sequence of the DE loop comprises SEQ ID NO: 96, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 86, thesequence of the DE loop consists of SEQ ID NO: 96, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:87, the sequence of the DE loop comprises SEQ ID NO: 97, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 87, thesequence of the DE loop consists of SEQ ID NO: 97, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:88, the sequence of the DE loop comprises SEQ ID NO: 95, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 88, thesequence of the DE loop consists of SEQ ID NO: 95, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:89, the sequence of the DE loop comprises SEQ ID NO: 94, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 89, thesequence of the DE loop consists of SEQ ID NO: 94, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:90, the sequence of the DE loop comprises SEQ ID NO: 94, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 90, thesequence of the DE loop consists of SEQ ID NO: 94, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:91, the sequence of the DE loop comprises SEQ ID NO: 95, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 91, thesequence of the DE loop consists of SEQ ID NO: 95, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:92, the sequence of the DE loop comprises SEQ ID NO: 98, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 92, thesequence of the DE loop consists of SEQ ID NO: 98, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:93, the sequence of the DE loop comprises SEQ ID NO: 94, and thesequence of the FG loop comprises SEQ ID NO: 9 or 139. In otherembodiments, the sequence of the BC loop consists of SEQ ID NO: 93, thesequence of the DE loop consists of SEQ ID NO: 94, and the sequence ofthe FG loop consists of SEQ ID NO: 9 or 139.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:168, the sequence of the DE loop comprises SEQ ID NO: 169, and thesequence of the FG loop comprises SEQ ID NO: 170. In other embodiments,the sequence of the BC loop consists of SEQ ID NO: 168, the sequence ofthe DE loop consists of SEQ ID NO: 169, and the sequence of the FG loopconsists of SEQ ID NO: 170.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:100, the sequence of the DE loop comprises SEQ ID NO: 118, and thesequence of the FG loop comprises SEQ ID NO: 129. In other embodiments,the sequence of the BC loop consists of SEQ ID NO: 100, the sequence ofthe DE loop consists of SEQ ID NO: 118, and the sequence of the FG loopconsists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 101, the sequenceof the DE loop comprises SEQ ID NO: 119, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 101, the sequence of the DE loop consists of SEQ ID NO: 119,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 102, the sequenceof the DE loop comprises SEQ ID NO: 120, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 102, the sequence of the DE loop consists of SEQ ID NO: 120,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 103, the sequenceof the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 103, the sequence of the DE loop consists of SEQ ID NO: 121,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 104, the sequenceof the DE loop comprises SEQ ID NO: 122, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 104, the sequence of the DE loop consists of SEQ ID NO: 122,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 105, the sequenceof the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 105, the sequence of the DE loop consists of SEQ ID NO: 121,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 106, the sequenceof the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 106, the sequence of the DE loop consists of SEQ ID NO: 123,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 107, the sequenceof the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 107, the sequence of the DE loop consists of SEQ ID NO: 123,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 108, the sequenceof the DE loop comprises SEQ ID NO: 118, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 108, the sequence of the DE loop consists of SEQ ID NO: 118,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 109, the sequenceof the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 109, the sequence of the DE loop consists of SEQ ID NO: 123,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 110, the sequenceof the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 110, the sequence of the DE loop consists of s SEQ ID NO:121, and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 111, the sequenceof the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loopcomprises SEQ ID NO: 130. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 111, the sequence of the DE loop consists of SEQ ID NO: 123,and the sequence of the FG loop consists of SEQ ID NO: 130.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 108, the sequenceof the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 108, the sequence of the DE loop consists of SEQ ID NO: 121,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 112, the sequenceof the DE loop comprises SEQ ID NO: 124, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 112, the sequence of the DE loop consists of SEQ ID NO: 124,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 113, the sequenceof the DE loop comprises SEQ ID NO: 125, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 113, the sequence of the DE loop consists of SEQ ID NO: 125,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 114, the sequenceof the DE loop comprises SEQ ID NO: 118, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 114, the sequence of the DE loop consists of SEQ ID NO: 118,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 115, the sequenceof the DE loop comprises SEQ ID NO: 126, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 115, the sequence of the DE loop consists of SEQ ID NO: 126,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 116, the sequenceof the DE loop comprises SEQ ID NO: 127, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 116, the sequence of the DE loop consists of SEQ ID NO: 127,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the AB loop comprises SEQ ID NO:136, the sequence of the BC loop comprises SEQ ID NO: 117, the sequenceof the DE loop comprises SEQ ID NO: 128, and the sequence of the FG loopcomprises SEQ ID NO: 129. In other embodiments, the sequence of the ABloop consists of SEQ ID NO: 136, the sequence of the BC loop consists ofSEQ ID NO: 117, the sequence of the DE loop consists of SEQ ID NO: 128,and the sequence of the FG loop consists of SEQ ID NO: 129.

In some embodiments, the sequence of the BC loop comprises SEQ ID NO:174, the sequence of the DE loop comprises SEQ ID NO: 175, and thesequence of the FG loop comprises SEQ ID NO: 177. In other embodiments,the sequence of the BC loop consists of SEQ ID NO: 174, the sequence ofthe DE loop consists of SEQ ID NO: 175, and the sequence of the FG loopconsists of SEQ ID NO: 177.

In some embodiments, the CD40L-specific monomer subunit comprises asequence selected from the group consisting of SEQ ID NO: 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42 and 146. In other embodiments, theCD40L-specific monomer subunit consists of a sequence selected from thegroup consisting of SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42 and 146.

In some embodiments, the CD40L-specific monomer subunit comprises SEQ IDNO: 28 or 146. In other embodiments, the CD40L-specific monomer subunitconsists of SEQ ID NO: 28 or 146.

In some embodiments, the CD40L-specific Tn3 monomer subunits of theinvention comprise the amino acid sequence:

(SEQ ID NO: 167) IEVKDVTDTTALITWX₁DX₂X₃X₄X₅X₆X₇X₈CELTYGIKDVPGDRTTIDLWX₉HX₁₀AX₁₁YSIGNLKPDTEYEVSLICRX₁₂GDMSSNPAKETFTT

-   -   wherein:    -   (a) X₁ represents amino acid residue serine (S) or leucine (L);    -   (b) X₂ represents amino acid residue aspartic acid (D) or        glutamic acid (E);    -   (c) X₃ represents amino acid residue histidine (H), isoleucine        (I), valine (V), phenylalanine (F) or tryptophan (W);    -   (d) X₄ represents amino acid residue alanine (A), glycine (G),        glutamic acid (E) or aspartic acid (D);    -   (e) X₅ represents amino acid residue glutamic acid (E), leucine        (L), glutamine (Q), serine (S), aspartic acid (D) or asparagine        (N);    -   (f) X₆ represents amino acid residue phenylalanine (F) or        tyrosine (Y);    -   (g) X₇ represents amino acid residue isoleucine (I), valine (V),        histidine (H), glutamic acid (E) or aspartic acid (D);    -   (h) X₈ represents amino acid residue glycine (G), tryptophan (W)        or valine (V);    -   (i) X₉ represents amino acid residue tryptophan (W),        phenylalanine (F) or tyrosine (Y);    -   (j) X₁₀ represents amino acid residue serine (S), glutamine (Q),        methionine (M) or histidine (H);    -   (k) X₁₁ represents amino acid residue tryptophan (W) or        histidine (H); and,    -   (l) X₁₂ represents amino acid residue arginine (R) or serine        (S).

In some embodiments, the CD40L-specific Tn3 monomer subunits of theinvention consist of the amino acid sequence:

(SEQ ID NO: 167) IEVKDVTDTTALITWX₁DX₂X₃X₄X₅X₆X₇X₈CELTYGIKDVPGDRTTIDLWX₉HX₁₀AX₁₁YSIGNLKPDTEYEVSLICRX₁₂GDMSSNPAKETFTT

-   -   wherein:    -   (a) X₁ represents amino acid residue serine (S) or leucine (L);    -   (b) X₂ represents amino acid residue aspartic acid (D) or        glutamic acid (E);    -   (c) X₃ represents amino acid residue histidine (H), isoleucine        (I), valine (V), phenylalanine (F) or tryptophan (W);    -   (d) X₄ represents amino acid residue alanine (A), glycine (G),        glutamic acid (E) or aspartic acid (D);    -   (e) X₅ represents amino acid residue glutamic acid (E), leucine        (L), glutamine (Q), serine (S), aspartic acid (D) or asparagine        (N);    -   (f) X₆ represents amino acid residue phenylalanine (F) or        tyrosine (Y);    -   (g) X₇ represents amino acid residue isoleucine (I), valine (V),        histidine (H), glutamic acid (E) or aspartic acid (D);    -   (h) X₈ represents amino acid residue glycine (G), tryptophan (W)        or valine (V);    -   (i) X₉ represents amino acid residue tryptophan (W),        phenylalanine (F) or tyrosine (Y);    -   (j) X₁₀ represents amino acid residue serine (S), glutamine (Q),        methionine (M) or histidine (H);    -   (k) X₁₁ represents amino acid residue tryptophan (W) or        histidine (H); and,    -   (l) X₁₂ represents amino acid residue arginine (R) or serine        (S).

In some embodiments, the CD40L-specific monomer subunit comprises asequence selected from the group consisting of SEQ ID NO: 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.In some embodiments, the CD40L-specific monomer subunit consists of asequence selected from the group consisting of SEQ ID NO: 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

In some embodiments, the CD40L-specific Tn3 monomer subunits of theinvention comprise the amino acid sequence:

(SEQ ID NO: 171) IEVX₁DVTDTTALITWX₂X₃RSX₄X₅X₆X₇X₈X₉X₁₀CELX₁₁YGIKDVPGDRTTIDLX₁₂X₁₃X₁₄X₁₅YVHYSIGNLKPDTX₁₆YEVSLICLTTDGTYX₁₇NP AKETFTT

-   -   wherein:    -   (a) X₁ represents amino acid residue lysine (K) or glutamic acid        (E);    -   (b) X₂ represents amino acid residue threonine (T) or isoleucine        (I);    -   (c) X₃ represents amino acid residue asparagine (N) or alanine        (A);    -   (d) X₄ represents amino acid residue serine (S), leucine (L),        alanine (A), phenylalanine (F) or tyrosine (Y);    -   (e) X₅ represents amino acid residue tyrosine (Y), alanine (A),        glycine (G), valine (V), isoleucine (I) or serine (S);    -   (f) X₆ represents amino acid residue tyrosine (Y), serine (S),        alanine (A) or histidine (H);    -   (g) X₇ represents amino acid residue asparagine (N), aspartic        acid (D), histidine (H) or tyrosine (Y);    -   (h) X₈ represents amino acid residue leucine (L), phenylalanine        (F), histidine (H) or tyrosine (Y);    -   (i) X₉ represents amino acid residue histidine (H), proline (P),        serine (S), leucine (L) or aspartic acid (D);    -   (j) X₁₀ represents amino acid residue glycine (G), phenylalanine        (F), histidine (H) or tyrosine (Y);    -   (k) X₁₁ represents amino acid residue alanine (A) or threonine        (T);    -   (l) X₁₂ represents amino acid residue serine (S), asparagine        (N), glutamic acid (E), asparagine (R) or aspartic acid (D);    -   (m) X₁₃ represents amino acid residue serine (S), glutamine (Q),        threonine (T), asparagine (N) or alanine (A);    -   (n) X₁₄ represents amino acid residue proline (P), valine (V),        isoleucine (I) or alanine (A) or no amino acid;    -   (o) X₁₅ represents amino acid residue isoleucine (I) or no amino        acid;    -   (p) X₁₆ represents amino acid residue glutamic acid (E) or        lysine (K); and,    -   (q) X₁₇ represents amino acid residue serine (S) or asparagine        (N).

In some embodiments, the CD40L-specific Tn3 monomer subunits of theinvention consist of the amino acid sequence:

(SEQ ID NO: 171) IEVX₁DVTDTTALITWX₂X₃RSX₄X₅X₆X₇X₈X₉X₁₀CELX₁₁YGIKDVPGDRTTIDLX₁₂X₁₃X₁₄X₁₅YVHYSIGNLKPDTX₁₆YEVSLICLTTDGTYX₁₇NP AKETFTT

-   -   wherein:    -   (a) X₁ represents amino acid residue lysine (K) or glutamic acid        (E);    -   (b) X₂ represents amino acid residue threonine (T) or isoleucine        (I);    -   (c) X₃ represents amino acid residue asparagine (N) or alanine        (A);    -   (d) X₄ represents amino acid residue serine (S), leucine (L),        alanine (A), phenylalanine (F) or tyrosine (Y);    -   (e) X₅ represents amino acid residue tyrosine (Y), alanine (A),        glycine (G), valine (V), isoleucine (I) or serine (S);    -   (f) X₆ represents amino acid residue tyrosine (Y), serine (S),        alanine (A) or histidine (H);    -   (g) X₇ represents amino acid residue asparagine (N), aspartic        acid (D), histidine (H) or tyrosine (Y);    -   (h) X₈ represents amino acid residue leucine (L), phenylalanine        (F), histidine (H) or tyrosine (Y);    -   (i) X₉ represents amino acid residue histidine (H), proline (P),        serine (S), leucine (L) or aspartic acid (D);    -   (j) X₁₀ represents amino acid residue glycine (G), phenylalanine        (F), histidine (H) or tyrosine (Y);    -   (k) X₁₁ represents amino acid residue alanine (A) or threonine        (T);    -   (l) X₁₂ represents amino acid residue serine (S), asparagine        (N), glutamic acid (E), asparagine (R) or aspartic acid (D);    -   (m) X₁₃ represents amino acid residue serine (S), glutamine (Q),        threonine (T), asparagine (N) or alanine (A);    -   (n) X₁₄ represents amino acid residue proline (P), valine (V),        isoleucine (I) or alanine (A) or no amino acid;    -   (o) X₁₅ represents amino acid residue isoleucine (I) or no amino        acid;    -   (p) X₁₆ represents amino acid residue glutamic acid (E) or        lysine (K); and,    -   (q) X₁₇ represents amino acid residue serine (S) or asparagine        (N).

In some embodiments, a CD40L-specific monomer scaffold comprise a Tn3module wherein one or more of the beta strands comprise at least oneamino acid substitution except that the cysteine residues in the C and Fbeta strands (SEQ ID NOs: 13 or 14; and SEQ ID NO: 17, respectively) maynot be substituted.

The loops connecting the various beta strands of a CD40L-specificmonomer subunit can be randomized for length and/or sequence diversity.In one embodiment, a CD40L-specific monomer subunit has at least oneloop that is randomized for length and/or sequence diversity. In oneembodiment, at least one, at least two, at least three, at least four,at least five or at least six loops of a CD40L-specific monomer subunitare randomized for length and/or sequence diversity. In one embodiment,at least one loop of a CD40L-specific monomer subunit is kept constantwhile at least one additional loop is randomized for length and/orsequence diversity. In another embodiment, at least one, at least two,or all three of loops AB, CD, and EF are kept constant while at leastone, at least two, or all three of loops BC, DE, and FG are randomizedfor length or sequence diversity. In another embodiment, at least one,at least two, or at least all three of loops AB, CD, and EF arerandomized while at least one, at least two, or all three of loops BC,DE, and FG are randomized for length and/or sequence diversity. In stillanother embodiment, at least one, at least two, at least three of loops,at least 4, at least five, or all six of loops AB, CD, EF, BC, DE, andFG are randomized for length or sequence diversity.

In some embodiments, one or more residues within a loop are heldconstant while other residues are randomized for length and/or sequencediversity. In some embodiments, one or more residues within a loop areheld to a predetermined and limited number of different amino acidswhile other residues are randomized for length and/or sequencediversity. Accordingly, a CD40L-specific monomer subunit of theinvention can comprise one or more loops having a degenerate consensussequence and/or one or more invariant amino acid residues.

In one embodiment, the CD40L-specific monomer subunit of the inventioncomprises an AB loop which is randomized. In another embodiment, theCD40L-specific monomer subunit of the invention comprises a BC loopwhich is randomized. In one embodiment, the CD40L-specific monomersubunit of the invention comprises a CD loop which is randomized. In oneembodiment, the CD40L-specific monomer subunit of the inventioncomprises a DE loop which is randomized. In one embodiment, theCD40L-specific monomer subunit of the invention comprises an EF loopwhich is randomized.

In certain embodiments, the CD40L-specific monomer subunit of theinvention comprises a FG loop which is held to be at least one aminoacid residue shorter than the cognate FG loop of the third FnIII domainof human tenascin C and is further randomized at one or more positions.

In specific embodiments, at least one of loops BC, DE, and FG israndomized, wherein the A beta strand comprises SEQ ID NO: 10 or 11, theB beta strand comprises SEQ ID NO:12, the C beta strand comprises SEQ IDNO:13 or 14, the D beta strand comprises SEQ ID NO:15, the E beta strandcomprises SEQ ID NO:16, the F beta strand comprises SEQ ID NO:17, andthe G beta strand comprises SEQ ID NO:18, the AB loop comprises SEQ IDNO:4 or 136, the CD loop comprises SEQ ID NO:6 and the EF loop comprisesSEQ ID NO:8 or 137.

In other specific embodiments, at least one of loops AB, CD, and EF arerandomized, wherein the A beta strand comprises SEQ ID NO: 10 or 11, theB beta strand comprises SEQ ID NO:12, the C beta strand comprises SEQ IDNO:13 or 14, the D beta strand comprises SEQ ID NO:15, the E beta strandcomprises SEQ ID NO:16, the F beta strand comprises SEQ ID NO: 17, andthe G beta strand comprises SEQ ID NO:18, the BC loop comprises SEQ IDNO:5, the DE loop comprises SEQ ID NO:7 and the FG loop comprises SEQ IDNO:9 or 139.

Enhanced Scaffold Stability

The stability of Tn3 scaffolds of the invention may be increased by manydifferent approaches. In some embodiments, Tn3 scaffolds of theinvention can be stabilized by elongating the N- and/or C-terminalregions. The N- and/or C-terminal regions can be elongated by 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more than 10 amino acids. In other embodiments,the Tn3 scaffolds of the invention can be stabilized by introducing analteration that increases serum half-life, as described herein. In yetanother embodiment, the Tn3 scaffolds of the invention comprise anaddition, deletion or substitution of at least one amino acid residue tostabilize the hydrophobic core of the scaffold.

Tn3 scaffolds of the invention can be effectively stabilized byengineering non-natural disulfide bonds as disclosed in InternationalPatent Application No. PCT/US2011/032184. In some embodiments, scaffoldsof the invention comprise non-naturally occurring disulfide bonds, asdescribed in PCT Publication No: WO 2009/058379. A bioinformaticsapproach may be utilized to identify candidate positions suitable forengineering disulfide bonds.

In one embodiment, a Tn3 monomer subunit of the invention comprises atleast one, at least two, at least three, at least four, or at least fivenon-naturally occurring intramolecular disulfide bonds. In oneembodiment, a Tn3 monomer subunit of the invention comprises at leastone non-naturally occurring intramolecular disulfide bond, wherein saidat least one non-naturally occurring disulfide bond stabilizes themonomer. In yet another embodiment, Tn3 scaffolds of the inventioncomprise at least one non-naturally occurring disulfide bond, whereinthe bond is located between two distinct monomer or multimer Tn3scaffolds, i.e., the disulfide bond is an intermolecular disulfide bond.For example, a disulfide bond can link distinct scaffolds (for example,two CD40L-specific monomer scaffolds), a Tn3 scaffold and a linker, aTn3 scaffold and an Fc domain, or a Tn3 scaffold and an antibody orfragment thereof.

In some embodiments, Tn3 scaffolds of the invention comprise at leastone non-naturally occurring intermolecular disulfide bond that links aTn3 monomer subunit and an isolated heterologous moiety, a Tn3 monomersubunit and a heterologous moiety fused or conjugated to the same Tn3scaffold, or a Tn3 monomer subunit and a heterologous moiety fused orconjugated to a different Tn3 scaffold.

In some embodiments, Tn3 scaffolds of the invention comprise a disulfidebond that forms a Tn3 multimeric scaffold of at least 2, at least 3, atleast 4 or more monomer subunits.

In another embodiment, Tn3 scaffolds of the invention may comprise anelongation of the N and/or C terminal regions. In one embodiment, theTn3 scaffold of the invention comprises an alteration to increase serumhalf-life, as described herein. In yet another embodiment, the scaffoldsof the invention comprise an addition, deletion or substitution of atleast one amino acid residue to stabilize the hydrophobic core of thescaffold.

Stability Measurements

The stability of the Tn3 monomer subunits of the invention, isolated oras part of a multimeric Tn3 scaffold, can be readily measured bytechniques well known in the art, such as thermal (T_(m)) and chaotropicdenaturation (such as treatment with urea, or guanidine salts), proteasetreatment (such as treatment with thermolysin) or another art acceptedmethodology to determine protein stability. A comprehensive review oftechniques used to measure protein stability can be found, for examplein “Current Protocols in Molecular Biology” and “Current Protocols inProtein Science” by John Wiley and Sons. 2007.

Multimeric Tn3 Scaffolds

One aspect of the present invention provides multimeric Tn3 scaffoldscomprising at least two Tn3 monomer subunits of the invention joined intandem, and wherein at least one of the monomers is a CD40L-specificmonomer subunit. Such multimeric Tn3 scaffolds can be assembled inmultiple formats. In a specific aspect, the invention providesmultimeric Tn3 scaffolds, wherein at least two CD40L-specific monomersubunits are connected in tandem via a peptide linker. In someembodiments, the multimeric Tn3 scaffold exhibits an increase in thevalency and/or avidity of target binding, or other action of thetarget(s). In some embodiments, the increase in valency and/or avidityof target binding is accomplished when multiple monomer subunits bind tothe same target. In some embodiments, the increase in valency improves aspecific action on the target, such as increasing the dimerization of atarget protein.

In a specific embodiment, a multimeric Tn3 scaffold of the inventioncomprises at least two CD40L-specific monomer subunits connected intandem, wherein each CD40L-specific monomer subunit binds at least onetarget, and wherein each CD40L-specific monomer subunit comprises aplurality of beta strands linked to a plurality of loop regions, whereinat least one loop is a non-naturally occurring variant of the cognateloop in the parent Tn3 scaffold (SEQ ID NO: 3).

In one embodiment, multimeric Tn3 scaffolds are generated throughcovalent binding between CD40L-specific monomer subunits, for example,by directly linking the CD40L-specific monomer subunits, or by theinclusion of a linker, e.g., a peptide linker. In particular examples,covalently bonded Tn3 scaffolds are generated by constructing fusiongenes that encode the CD40L-specific monomer subunits or, alternatively,by engineering codons for cysteine residues into CD40L-specific monomersubunits and allowing disulfide bond formation to occur between theexpression products.

In one embodiment, multimeric Tn3 scaffolds of the invention comprise atleast two CD40L-specific monomer subunits that are connected directly toeach other without any additional intervening amino acids. In anotherembodiment, multimeric Tn3 scaffolds of the invention comprise at leasttwo CD40L-specific monomer subunits that are connected in tandem via alinker, e.g., a peptide linker.

In a specific embodiment, multimeric Tn3 scaffolds of the inventioncomprise at least two CD40L-specific monomer subunits that are connectedin tandem via a peptide linker, wherein the peptide linker comprises 1to about 1000, or 1 to about 500, or 1 to about 250, or 1 to about 100,or 1 to about 50, or 1 to about 25, amino acids. In a specificembodiment, the multimeric Tn3 scaffold comprises at least twoCD40L-specific monomer subunits that are connected in tandem via apeptide linker, wherein the peptide linker comprises 1 to about 20, or 1to about 15, or 1 to about 10, or 1 to about 5, amino acids.

In a specific embodiment, the multimeric Tn3 scaffold comprises at leasttwo CD40L-specific monomer subunits that are connected in tandem via alinker, e.g., a peptide linker, wherein the linker is a functionalmoiety. The functional moiety will be selected based on the desiredfunction and/or characteristics of the multimeric Tn3 scaffold. Forexample, a functional moiety useful for purification (e.g., a histidinetag) may be used as a linker. Functional moieties useful as linkersinclude, but are not limited to, polyethylene glycol (PEG), a cytotoxicagent, a radionuclide, imaging agent, biotin, a dimerization domain,human serum albumin (HSA) or an FcRn binding portion thereof, a domainor fragment of an antibody, a single chain antibody, a domain antibody,an albumin binding domain, an IgG molecule, an enzyme, a ligand, areceptor, a binding peptide, a non-Tn3 scaffold, an epitope tag, arecombinant polypeptide polymer, a cytokine, and the like. Specificpeptide linkers and functional moieties which may be used as linkers aredisclosed infra.

In specific embodiments, the functional moiety is an immunoglobulin or afragment thereof. In some embodiments, the immunoglobulin or fragmentthereof comprises an Fc domain. In some embodiments, the Fc domain failsto induce at least one FcγR-mediated effector function, such as ADCC(Antibody-dependent cell-mediated cytotoxicity). It is known in the artthat the Fc domain maybe altered to reduce or eliminate at least oneFcγR-mediated effector function, see, for example, U.S. Pat. Nos.5,624,821 and 6,737,056.

In some embodiments, the multimeric Tn3 scaffold comprises at least twoCD40L-specific monomer subunits that are connected via one or morelinkers, wherein the linkers interposed between each CD40L-specificmonomer subunit can be the same linkers or different linkers. In someembodiments, a linker can comprise multiple linkers, which can be thesame linker or different linkers. In some embodiments, when a pluralityof linkers are concatenated, some or all the linkers can be functionalmoieties.

Scaffold Binding Stoichiometry

In some embodiments, a monomeric or multimeric Tn3 scaffold can comprisea CD40L-specific monomer subunit specific for different epitopes, whichcan be different epitopes on a single CD40L molecule or on differentCD40L target molecules. In some embodiments, a multimeric Tn3 scaffoldcan comprise CD40L-specific monomer subunits wherein each subunittargets one or more different epitopes on one or more CD40L molecules.

In other embodiments, a monomeric or multimeric Tn3 scaffold can bindtwo or more different epitopes on the same CD40L molecule. In someembodiments, the different epitopes are non-overlapping epitopes. Inother embodiments, the different epitopes are overlapping epitopes.

In yet another specific embodiment, a monomeric or multimeric Tn3scaffold can bind one or more epitopes on a CD40L molecule andadditionally bind one or more epitopes on a second CD40L molecule. Insome embodiments, the different target molecules are part of anoligomeric complex, e.g., a trimeric CD40L complex.

In still another specific embodiment, a monomeric or multimeric Tn3scaffold can bind to a single epitope on a CD40L trimer. In yet anotherembodiment, a monomeric or multimeric Tn3 scaffold can bind to the sameepitope on at least two CD40L trimers.

In certain embodiments, a monomeric or multimeric Tn3 scaffold can bindthe same epitope on two or more copies of a CD40L molecule on anadjacent cell surface. In certain embodiments, a monomeric or multimericTn3 scaffold can bind the same epitope on two or more copies of a CD40Lmolecule in solution. In some embodiments, a monomeric or multimeric Tn3scaffold can bind to the same epitope or different epitopes on CD40Lwith the same or different binding affinities and/or avidities.

In another embodiment, a monomeric or multimeric Tn3 scaffolds can bindto epitopes on one or more copies of CD40L and achieve or enhance (e.g.,synergistically) a desired action on the target, e.g., prevent bindingto a receptor or prevent oligomerization.

In addition, when a monomeric or multimeric Tn3 scaffold of theinvention comprises a plurality of CD40L-specific monomer subunits,e.g., different monomers wherein each monomer targets different epitopeson CD40L, such monomer subunits can be arranged according to a certainpattern or special orientation to achieve or enhance a certainbiological effect. Such combinations of monomeric subunits can beassembled and subsequently evaluated using methods known in the art.

Fusions

The invention provides Tn3 scaffolds wherein at least one CD40L-specificmonomer subunit can be fused to a heterologous moiety. In this contextthe heterologous moiety is not used to link the scaffolds as a spacerbut may provide additional functionality to the Tn3 scaffold. In someembodiments, a heterologous moiety can also function as a linker. Thepresent invention encompasses the use of Tn3 scaffolds conjugated orfused to one or more heterologous moieties, including but not limitedto, peptides, polypeptides, proteins, fusion proteins, nucleic acidmolecules, small molecules, mimetic agents, synthetic drugs, inorganicmolecules, and organic molecules. Accordingly, the invention providespolypeptides comprising one or more CD40L-specific Tn3 monomer,including but not limited to the fusion proteins described herein.

The present invention encompasses the use of Tn3 scaffolds recombinantlyfused or chemically conjugated to a heterologous protein or polypeptideor fragment thereof. Conjugation includes both covalent and non-covalentconjugation. In some embodiments, a Tn3 scaffold can be fused orchemically conjugated to a polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 200, at least 300, at least 500,or at least 1000 amino acids) to generate fusion proteins.

The fusion or conjugation of a Tn3 scaffold to one or more heterologousmoieties can be direct, i.e., without a linker interposed between a Tn3scaffold and a heterologous moiety, or via one or more linker sequencesdescribed herein. In some embodiments, scaffolds can be used to targetheterologous polypeptides to particular cell types, either in vitro orin vivo, by fusing or conjugating the Tn3 scaffolds to antibodiesspecific for particular cell surface receptors in the target cells.

Tn3 scaffolds fused or conjugated to heterologous polypeptides can alsobe used in in vitro immunoassays and purification methods using methodsknown in the art. See, e.g., International Publication No. WO 93/21232;European Patent No. EP 439,095; Naramura et al., Immunol. Lett.39:91-99, 1994; U.S. Pat. No. 5,474,981; Gillies et al., PNAS89:1428-1432, 1992; and Fell et al., J. Immunol. 146:2446-2452, 1991,which are incorporated by reference in their entireties.

In some embodiments, the Tn3 scaffolds can be integrated with the humanimmune response by fusing or conjugating a scaffold with animmunoglobulin or domain thereof including, but not limited to, theconstant region of an IgG (Fc), e.g., through the N or C-terminus.Similarly, a fusion between a Tn3 scaffold and a complement protein,such as CIq, can be used to target cells.

Various publications describe methods for obtaining physiologicallyactive molecules whose half-lives are modified by introducing anFcRn-binding polypeptide into the molecules (see, e.g., WO 97/43316;U.S. Pat. Nos. 5,869,046; 5,747,035; WO 96/32478; and WO 91/14438), byfusing the molecules with antibodies whose FcRn-binding affinities arepreserved but affinities for other Fc receptors have been greatlyreduced (see, e.g., WO 99/43713), or by fusing the molecules with FcRnbinding domains of antibodies (see, e.g., WO 00/09560; U.S. Pat. No.4,703,039). Specific techniques and methods of increasing half-life ofphysiologically active molecules can also be found in U.S. Pat. No.7,083,784. Specifically, it is contemplated that the Tn3 scaffolds canbe fused to an Fc region from an IgG, wherein the Fc region comprisesamino acid residue mutations M252Y/S254T/T256E or H433K/N434F/Y436H,wherein amino acid positions are designated according to the Kabatnumbering schema. It is specifically contemplated the fusion of a Tn3scaffold to an Fc domain variant not capable of inducing ADCC.

In some embodiments, the half-life of the Tn3 scaffold can be increasedby genetically fusing the Tn3 scaffold with an intrinsicallyunstructured recombinant polypeptide (e.g., an XTEN™ polypeptide) or byconjugation with polyethylene glycol (PEG).

In some embodiments, the Tn3 scaffold can be fused with molecules thatincrease or extend in vivo or serum half-life. In some embodiments, thescaffold can be fused or conjugated with albumin, such as human serumalbumin (HSA), a neonatal Fc receptor (FcRn) binding fragment thereof,PEG, polysaccharides, antibodies, complement, hemoglobin, a bindingpeptide, lipoproteins and other factors to increase its half-life in thebloodstream and/or its tissue penetration. Any of these fusions may begenerated by standard techniques, for example, by expression of thefusion protein from a recombinant fusion gene constructed using publiclyavailable gene sequences.

In some embodiments, a property of the Tn3 scaffold can be improved byconjugation or fusion to an HSA variant, i.e., a molecule derived fromfull length HSA (SEQ ID NO: 139) comprising at least an amino acidsubstitution, a deletion, or a sequence truncation.

In some embodiments, the property improved by conjugation with an HSAvariant is plasma half-life. The improvement in plasma half-life of theTn3 scaffold can be an alteration in that property such as an increaseor decrease in plasma half-life, or changes in other pharmacokineticparameters. In some embodiments, the HSA variant is a mutant derivedfrom full length HSA (SEQ ID NO: 138). In a specific embodiment, the HSAvariant comprises a substitution of cysteine at position 34 to serine(SEQ ID NO: 133). HSA variants that can be used to modify the plasmahalf-life of a Tn3 scaffold are described, e.g., in InternationalPublications WO 2011/103076 and WO 2011/051489, both of which areincorporated by reference in their entireties. In some embodiments, theplasma half-life of a Tn3 scaffold of the invention is increased byfusing it with an HSA variant comprising at least one amino acidsubstitution in domain III of HSA.

In some embodiments, the Tn3 scaffold of the invention comprises an HSAvariant comprising the sequence of full-length mature HSA (SEQ ID NO:138) or a fragment thereof, except for at least one amino acidsubstitution, numbered relative to the position in full length matureHSA, at a position selected from the group consisting of 407, 415, 463,500, 506, 508, 509, 511, 512, 515, 516, 521, 523, 524, 526, 535, 550,557, 573, 574, and 580; wherein the at least one amino acid substitutiondoes not comprise a lysine (K) to glutamic acid (E) at position 573, andwherein the Tn3 scaffold has a plasma half-life longer than the plasmahalf-life of a Tn3 scaffold not conjugated to the HSA variant.

In some other embodiments, at least one amino acid substitution,numbered relative to the position in full length mature HSA, is at aposition selected from the group consisting of 463, 508, 523, and 524,wherein the Tn3 scaffold has a plasma half-life longer than the plasmahalf-life of a Tn3 scaffold not conjugated to the HSA variant.

In other embodiments, a Tn3 scaffold of the invention comprises an HSAvariant comprising the sequence of full-length mature HSA (SEQ ID NO:133 or 138) or a fragment thereof, except for at least one amino acidsubstitution, numbered relative to the position in full length matureHSA, selected from the group consisting of:

-   -   (a) substitution of Leucine (L) at position 407 to        Asparagine (N) or Tyrosine (Y);    -   (b) substitution of Valine (V) at position 415 to Threonine (T);    -   (c) substitution of Leucine (L) at position 463 to Asparagine        (N);    -   (d) substitution of Lysine (K) at position 500 to Arginine (R);    -   (e) substitution of Threonine (T) at position 506 to Tyrosine        (Y);    -   (f) substitution of Threonine (T) at position 508 to Arginine        (R);    -   (g) substitution of Phenylalanine (F) at position 509 to        Methionine (M) or Tryptophan (W);    -   (h) substitution of Alanine (A) at position 511 to Phenylalanine        (F);    -   (i) substitution of Aspartic Acid (D) at position 512 to        Tyrosine (Y);    -   (j) substitution of Threonine (T) at position 515 to Glutamine        (Q);    -   (k) substitution of Leucine (L) at position 516 to Threonine (T)        or Tryptophan (W);    -   (l) substitution of Arginine (R) at position 521 to Tryptophan        (W);    -   (m) substitution of Isoleucine (I) at position 523 to Aspartic        Acid (D), Glutamic Acid (E), Glycine (G), Lysine (K), or        Arginine (R);    -   (n) substitution of Lysine (K) at position 524 to Leucine (L);    -   (o) substitution of Glutamine (Q) at position 526 to Methionine        (M);    -   (p) substitution of Histidine (H) at position 535 to Proline        (P);    -   (q) substitution of Aspartic Acid (D) at position 550 to        Glutamic Acid (E);    -   (r) substitution of Lysine (K) at position 557 to Glycine (G);    -   (s) substitution of Lysine (K) at position 573 to Phenylalanine        (F), Histidine (H), Proline (P), Tryptophan (W), or Tyrosine        (Y);    -   (t) substitution of Lysine (K) at position 574 to Asparagine        (N);    -   (u) substitution of Glutamine (Q) at position 580 to Lysine (K);        and,    -   (v) a combination of two or more of said substitutions,    -   wherein said Tn3 scaffold has a plasma half-life longer than the        plasma half-life of a Tn3 scaffold not conjugated to said HSA        variant.

In some embodiments, the Tn3 scaffold comprises a HSA variant whichcomprises the sequence of full-length mature HSA (SEQ ID NO: 133 or 138)or a fragment thereof, except for at least one amino acid substitution,numbered relative to the position in full length mature HSA, selectedfrom the group consisting of:

-   -   (a) substitution of Leucine (L) at position 463 to Asparagine        (N);    -   (b) substitution of Threonine (T) at position 508 to Arginine        (R);    -   (c) substitution of Isoleucine (I) at position 523 to Aspartic        Acid (D), Glutamic Acid (E), Glycine (G), Lysine (K), or        Arginine (R);    -   (d) substitution of Lysine (K) at position 524 to Leucine (L);        and,    -   (e) a combination of two or more of said substitutions,    -   wherein said Tn3 scaffold has a plasma half-life longer than the        plasma half-life of a Tn3 scaffold not conjugated to said HSA        variant.

Moreover, the Tn3 scaffolds of the invention can be fused to markersequences, such as a peptide to facilitate purification. In someembodiments, the marker amino acid sequence is a poly-histidine peptide(His-tag), e.g., a octa-histidine-tag (His-8-tag) or hexa-histidine-tag(His-6-tag) such as the tag provided in a pQE expression vector (QIAGEN,Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among other vectors,many of which are commercially available. As described in Gentz et al.,Proc. Natl. Acad. Sci. USA 86:821-824, 1989, for instance,poly-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, a hemagglutinin (“HA”) tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (see, e.g., Wilson etal., Cell 37:767, 1984), a FLAG tag, a Strep-tag, a myc-tag, a V5 tag, aGFP-tag, an AU1-tag, an AU5-tag, an ECS-tag, a GST-tag, or an OLLAS tag.

Additional fusion proteins comprising Tn3 scaffolds of the invention maybe generated through the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”).

DNA shuffling may be employed to alter the action of Tn3 scaffolds onthe target (e.g., generate scaffolds with higher affinities and lowerdissociation rates). Tn3 scaffolds may be altered by random mutagenesisby error-prone PCR, random nucleotide insertion, or other methods priorto recombination. One or more portions of a polynucleotide encoding ascaffold, which bind to a specific target may be recombined with one ormore components, motifs, sections, parts, domains, fragments, etc. ofone or more heterologous molecules.

Antibody and Fc Domain Fusions

In some embodiments, the Tn3 scaffold of the invention comprises aCD40L-specific monomer subunit fused to a domain or fragment of anantibody (e.g., an IgG), including, but not limited to, an Fc domain.

In some embodiments, only one CD40L-specific monomer subunit isconjugated or fused to a domain or fragment of an antibody. Forinstance, a single a CD40L-specific monomer subunit can be fused to theN-terminus of a polypeptide of a domain or fragment of an antibody(e.g., a heavy chain or a light chain of an antibody). In otherembodiments, Tn3 scaffolds are created by fusing or conjugating one ormore CD40L-specific monomer subunits to the N-terminus and/or theC-terminus a polypeptide of a domain or fragment of an antibody (e.g., aheavy chain and/or a light chain of an antibody, or an Fc domain).

In some embodiments, some or all the a CD40L-specific monomer subunitsfused to a domain or fragment of an antibody are identical. In someother embodiments, some or all the a CD40L-specific monomer subunitfused to a domain or fragment of an antibody are different.

In a specific embodiment, the Tn3 scaffold of the invention comprisesone CD40L-specific monomer subunit fused to an Fc domain. In otherembodiments, the Tn3 scaffold of the invention comprises at least twoCD40L-specific monomer subunits fused to an Fc domain. In one specificembodiment, two of the CD40L-specific monomer subunits fused to an Fcdomain are identical. In one specific embodiment, two of theCD40L-specific monomer subunits fused to an Fc domain are different. Inone specific embodiment, two CD40L-specific monomer subunits fused to anFc domain are connected to each other in tandem, and one of theCD40L-specific monomer subunits is fused to the Fc domain.

In some embodiments, different Tn3 scaffolds of the invention can bedimerized by the use of Fc domain mutations which favor the formation ofheterodimers. It is known in the art that variants of the Fc region(e.g., amino acid substitutions and/or additions and/or deletions)enhance or diminish effector function of the antibody and can alter thepharmacokinetic properties (e.g. half-life) of the antibody. Thus, incertain embodiments, the Tn3 scaffolds of the invention comprise Fcdomain(s) that comprise an altered Fc region in which one or morealterations have been made in the Fc region in order to changefunctional and/or pharmacokinetic properties of the Tn3 scaffold. Incertain embodiments, the Tn3 scaffolds of the invention comprise Fcdomain(s) that comprise an altered Fc region in which one or morealterations have been made in the Fc region in order reduce or eliminateat least one Fc R-mediated effector function.

It is also known that the glycosylation of the Fc region can be modifiedto increase or decrease effector function and/or anti-inflammatoryactivity. Accordingly, in one embodiment a Tn3 scaffold of the inventioncomprise an Fc region with altered glycosylation of amino acid residuesin order to change cytotoxic and/or anti-inflammatory properties of theTn3 scaffolds.

Tn3 Scaffold Topologies

The Tn3 scaffolds of the invention can be fused to the C-terminus of theFc domains, antibody light chains, and antibody heavy chains in anysuitable spatial arrangement. See, e.g., International PublicationPCT/US2011/032184 for a detailed description of contemplated scaffoldtopologies.

Generation of Scaffolds of the Invention

The Tn3 scaffolds described herein may be used in any technique forevolving new or improved target binding proteins. In one particularexample, the target is immobilized on a solid support, such as a columnresin or microtiter plate well, and the target contacted with a libraryof candidate scaffold-based binding proteins. Such a library may consistof clones constructed from a Tn3 scaffold, through randomization of thesequence and/or the length of the CDR-like loops.

In this regard, bacteriophage (phage) display is one well knowntechnique which allows one to screen large oligopeptide libraries toidentify member(s) of those libraries which are capable of specificallybinding to a target. Phage display is a technique by which variantpolypeptides are displayed as fusion proteins to the coat protein on thesurface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990)Science 249: 386). A bioinformatics approach may be employed todetermine the loop length and diversity preferences of naturallyoccurring FnIII domains. Using this analysis, the preferences for looplength and sequence diversity may be employed to develop a “restrictedrandomization” approach. In this restricted randomization, the relativeloop length and sequence preferences are incorporated into thedevelopment of a library strategy. Integrating the loop length andsequence diversity analysis into library development results in arestricted randomization (i.e. certain positions within the randomizedloop are limited in which amino acid could reside in that position).

The invention also provides recombinant libraries comprising diversepopulations of non-naturally occurring Tn3 scaffolds. In one embodiment,the libraries comprise non-naturally occurring Tn3 scaffolds comprising,a plurality of beta strand domains linked to a plurality of loopregions, wherein one or more of said loops vary by deletion,substitution or addition by at least one amino acid. In a specificembodiment, the libraries comprise Tn3 scaffolds derived from the wildtype Tn3 scaffold.

As detailed above, the loops connecting the various beta strands of thescaffolds may be randomized for length and/or sequence diversity. In oneembodiment, the libraries of the invention comprise Tn3 scaffolds havingat least one loop that is randomized for length and/or sequencediversity. In one embodiment, at least one, at least two, at leastthree, at least four, at least five or at least six loops of the Tn3scaffolds are randomized for length and/or sequence diversity. In oneembodiment, at least one loop is kept constant while at least oneadditional loop is randomized for length and/or sequence diversity. Inanother embodiment, at least one, at least two, or all three of loopsAB, CD, and EF are kept constant while at least one, at least two, orall three of loops BC, DE, and FG are randomized for length or sequencediversity. In another embodiment, at least one, at least two, or atleast all three of loops AB, CD, and EF are randomized while at leastone, at least two, or all three of loops BC, DE, and FG are randomizedfor length and/or sequence diversity.

In a specific embodiment, the libraries of the invention comprise FnIIIscaffolds, wherein the A beta strand comprises SEQ ID NO: 10 or 11, theB beta strand comprises SEQ ID NO: 12, the C beta strand comprises SEQID NO: 13 or 14, the D beta strand comprises SEQ ID NO: 15, the E betastrand comprises SEQ ID NO: 16, the F beta strand comprises SEQ ID NO:17, and the G beta strand comprises SEQ ID NO: 18.

In a specific embodiment, the libraries of the invention comprise FnIIIscaffolds, wherein the A beta strand consists of SEQ ID NO: 10 or 11,the B beta strand consists of SEQ ID NO: 12, the C beta strand consistsof SEQ ID NO: 13 or 14, the D beta strand consists of SEQ ID NO: 15, theE beta strand consists of SEQ ID NO: 16, the F beta strand consists ofSEQ ID NO: 17, and the G beta strand consists of SEQ ID NO: 18.

In a specific embodiment, the libraries of the invention comprise FnIIIscaffolds, wherein the A beta strand consists essentially of SEQ ID NO:10 or 11, the B beta strand consists essentially of SEQ ID NO: 12, the Cbeta strand consists essentially of SEQ ID NO: 13 or 14, the D betastrand consists essentially of SEQ ID NO: 15, the E beta strand consistsessentially of SEQ ID NO: 16, the F beta strand consists essentially ofSEQ ID NO: 17, and the G beta strand consists essentially of SEQ ID NO:18.

As detailed above, one or more residues within a loop may be heldconstant while other residues are randomized for length and/or sequencediversity. Optionally or alternatively, one or more residues within aloop may be held to a predetermined and limited number of differentamino acids while other residues are randomized for length and/orsequence diversity. Accordingly, libraries of the invention comprise Tn3scaffolds that may comprise one or more loops having a degenerateconsensus sequence and/or one or more invariant amino acid residues. Inanother embodiment, the libraries of the invention comprise Tn3scaffolds having BC loops which are randomized. In another embodiment,the libraries of the invention comprise Tn3 scaffolds having BC loopswhich are randomized. In still another embodiment, the libraries of theinvention comprise Tn3 scaffolds having BC loops which are randomized.

In one embodiment the libraries of the invention comprise Tn3 scaffoldshaving DE loops which are randomized. In one embodiment, the librariesof the invention comprise Tn3 scaffolds having FG loops which arerandomized. In another embodiment, the libraries of the inventioncomprise FnIII scaffolds having FG loops which are randomized.

In a specific embodiment, the libraries of the invention comprisescaffolds, wherein the scaffolds comprise the amino acid sequence:

(SEQ ID NO: 11) IEV (SEQ ID NO: 12) (X_(AB))_(n)ALITW (SEQ ID NO: 173)(X_(BC))_(n)CELX1YGI (SEQ ID NO: 15) (X_(CD))_(n)TTIDL (SEQ ID NO: 16)(X_(DE))_(n)YSI (SEQ ID NO: 17) (X_(EF))_(n)YEVSLIC (SEQ ID NO: 18)(X_(FG))_(n)KETFTT

-   -   wherein:    -   (a) X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent        the amino acid residues present in the sequences of the AB, BC,        CD, DE, EF, and FG loops, respectively;    -   (b) X₁ represents amino acid residue A or T; and,    -   (c) length of the loop n is an integer between 2 and 26.

In some embodiments, the libraries of the invention compriseCD40L-specific Tn3 monomer subunits of the invention comprising theamino acid sequence:

(SEQ ID NO: 167) IEVKDVTDTTALITWX₁DX₂X₃X₄X₅X₆X₇X₈CELTYGIKDVPGDRTTIDLWX₉HX₁₀AX₁₁YSIGNLKPDTEYEVSLICRX₁₂GDMSSNPAKETFTT

-   -   wherein:    -   (a) X₁ represents amino acid residue serine (S) or leucine (L);    -   (b) X₂ represents amino acid residue aspartic acid (D) or        glutamic acid (E);    -   (c) X₃ represents amino acid residue histidine (H), isoleucine        (I), valine (V), phenylalanine (F) or tryptophan (W);    -   (d) X₄ represents amino acid residue alanine (A), glycine (G),        glutamic acid (E) or aspartic acid (D);    -   (e) X₅ represents amino acid residue glutamic acid (E), leucine        (L), glutamine (Q), serine (S), aspartic acid (D) or asparagine        (N);    -   (f) X₆ represents amino acid residue phenylalanine (F) or        tyrosine (Y);    -   (g) X₇ represents amino acid residue isoleucine (I), valine (V),        histidine (H), glutamic acid (E) or aspartic acid (D);    -   (h) X₈ represents amino acid residue glycine (G), tryptophan (W)        or valine (V);    -   (i) X₉ represents amino acid residue tryptophan (W),        phenylalanine (F) or tyrosine (Y);    -   (j) X₁₀ represents amino acid residue serine (S), glutamine (Q),        methionine (M) or histidine (H);    -   (k) X₁₁ represents amino acid residue tryptophan (W) or        histidine (H); and,    -   (l) X₁₂ represents amino acid residue arginine (R) or serine        (S).

In some embodiments, the libraries of the invention compriseCD40L-specific Tn3 monomer subunits of the invention comprising theamino acid sequence:

(SEQ ID NO: 171) IEVX₁DVTDTTALITWX₂X₃RSX₄X₅X₆X₇X₈X₉X₁₀CELX₁₁YGIKDVPGDRTTIDLX₁₂X₁₃X₁₄X₁₅YVHYSIGNLKPDTX₁₆YEVSLICLTTDGTYX₁₇NP AKETFTT

-   -   wherein:    -   (a) X₁ represents amino acid residue lysine (K) or glutamic acid        (E);    -   (b) X₂ represents amino acid residue threonine (T) or isoleucine        (I);    -   (c) X₃ represents amino acid residue asparagine (N) or alanine        (A);    -   (d) X₄ represents amino acid residue serine (S), leucine (L),        alanine (A), phenylalanine (F) or tyrosine (Y);    -   (e) X₅ represents amino acid residue tyrosine (Y), alanine (A),        glycine (G), valine (V), isoleucine (I) or serine (S);    -   (f) X₆ represents amino acid residue tyrosine (Y), serine (S),        alanine (A) or histidine (H);    -   (g) X₇ represents amino acid residue asparagine (N), aspartic        acid (D), histidine (H) or tyrosine (Y);    -   (h) X₈ represents amino acid residue leucine (L), phenylalanine        (F), histidine (H) or tyrosine (Y);    -   (i) X₉ represents amino acid residue histidine (H), proline (P),        serine (S), leucine (L) or aspartic acid (D);    -   (j) X₁₀ represents amino acid residue glycine (G), phenylalanine        (F), histidine (H) or tyrosine (Y);    -   (k) X₁₁ represents amino acid residue alanine (A) or threonine        (T);    -   (l) X₁₂ represents amino acid residue serine (S), asparagine        (N), glutamic acid (E), asparagine (R) or aspartic acid (D);    -   (m) X₁₃ represents amino acid residue serine (S), glutamine (Q),        threonine (T), asparagine (N) or alanine (A);    -   (n) X₁₄ represents amino acid residue proline (P), valine (V),        isoleucine (I) or alanine (A) or no amino acid;    -   (o) X₁₅ represents amino acid residue isoleucine (I) or no amino        acid;    -   (p) X₁₆ represents amino acid residue glutamic acid (E) or        lysine (K); and,    -   (q) X₁₇ represents amino acid residue serine (S) or asparagine        (N).

The invention further provides methods for identifying a recombinant Tn3scaffold that binds a target, e.g., CD40L, and has increased stabilityor improved action on the target, e.g., CD40L, as compared to a parentTn3 scaffold by screening the libraries of the invention.

In certain embodiments, the method for identifying a recombinant Tn3scaffold having increased protein stability as compared to a parent Tn3scaffold, and which specifically binds a target, comprises:

contacting the target ligand with a library of the invention underconditions suitable for forming a scaffold:target ligand complex;

obtaining from the complex, the scaffold that binds the target ligand;

determining if the stability of the scaffold obtained in step (b) isgreater than that of the wild type Tn3 scaffold.

The same method can be used to identify a recombinant Tn3 scaffold withimproved binding affinity, avidity, etc. to the target. In oneembodiment, in step (a) the scaffold library of the invention isincubated with immobilized target. In one embodiment, in step (b) thescaffold:target ligand complex is washed to remove non-specific binders,and the tightest binders are eluted under very stringent conditions andsubjected to PCR to recover the sequence information. It is specificallycontemplated that the binders and/or sequence information obtained instep (b) can be used to create a new library using the methods disclosedherein or known to one of skill in the art, which may be used to repeatthe selection process, with or without further mutagenesis of thesequence. In some embodiments, a number of rounds of selection may beperformed until binders of sufficient affinity for the antigen areobtained.

A further embodiment of the invention is a collection of isolatednucleic acid molecules encoding a library comprising the scaffolds ofthe invention and as described above.

The scaffolds of the invention may be subjected to affinity maturation.In this art-accepted process, a specific binding protein is subject to ascheme that selects for increased affinity for a specific target (see Wuet al., Proc. Natl. Acad. Sci. USA. 95(11):6037-42). The resultantscaffolds of the invention may exhibit binding characteristics at leastas high as compared to the scaffolds prior to affinity maturation.

The invention also provides methods of identifying the amino acidsequence of a protein scaffold capable of binding to target so as toform a scaffold:target complex. In one embodiment, the method comprises:(a) contacting a library of the invention with an immobilized orseparable target; (b) separating the scaffold:target complexes from thefree scaffolds; (c) causing the replication of the separated scaffoldsof (b) so as to result in a new polypeptide display librarydistinguished from that in (a) by having a lowered diversity and bybeing enriched in displayed scaffolds capable of binding the target; d)optionally repeating steps (a), and (b) with the new library of (c); ande) determining the nucleic acid sequence of the region encoding thedisplayed scaffold of a species from (d) and hence deducing the peptidesequence capable of binding to the target.

In another embodiment, the Tn3 scaffolds of the invention may be furtherrandomized after identification from a library screen. In oneembodiment, methods of the invention comprise further randomizing atleast one, at least two, at least three, at least four, at least five orat least six loops of a scaffold identified from a library using amethod described herein. In another embodiment, the further randomizedscaffold is subjected to a subsequent method of identifying a scaffoldcapable of binding a target. This method comprises (a) contacting saidfurther randomized scaffold with an immobilized or separable target, (b)separating the further randomized scaffold:target complexes from thefree scaffolds, (c) causing the replication of the separated scaffoldsof (b), optionally repeating steps (a)-(c), and (d) determining thenucleic acid sequence of the region encoding said further randomizedscaffold and hence, deducing the peptide sequence capable of binding tothe target.

In a further embodiment, the further randomized scaffolds comprise atleast one, at least two, at least three, at least four, at least five,or at least six randomized loops which were previously randomized in thefirst library. In an alternate further embodiment, the furtherrandomized scaffolds comprise at least one, at least two, at leastthree, at least four, at least five, or at least six randomized loopswhich were not previously randomized in the first library.

The invention also provides a method for obtaining at least two Tn3scaffolds that bind to at least one or more targets. This method allowsfor the screening of agents that act cooperatively to elicit aparticular response. It may be advantageous to use such a screen when anagonistic activity requiring the cooperation of more than one scaffoldis required. This method allows for the screening of cooperative agentswithout the reformatting of the library to form multimeric complexes. Inone embodiment, the method of the invention comprises contacting atarget ligand with a library of the invention under conditions thatallow a scaffold:target ligand complex to form, engaging said scaffoldswith a crosslinking agent (defined as an agent that brings together, inclose proximity, at least two identical or distinct scaffolds) whereinthe crosslinking of the scaffolds elicits a detectable response andobtaining from the complex, said scaffolds that bind the target. In afurther embodiment, the crosslinking agent is a scaffold specificantibody, or fragment thereof, an epitope tag specific antibody of afragment thereof, a dimerization domain, such as Fc region, a coiledcoil motif (for example, but not limited to, a leucine zipper), achemical crosslinker, or another dimerization domain known in the art.

The invention also provides methods of detecting a compound utilizingthe Tn3 scaffolds of the invention. Based on the binding specificitiesof the Tn3 scaffolds obtained by library screening, it is possible touse such Tn3 scaffolds in assays to detect the specific target in asample, such as for diagnostic methods. In one embodiment, the method ofdetecting a compound comprises contacting said compound in a sample witha Tn3 scaffold of the invention, under conditions that allow acompound:scaffold complex to form and detecting said scaffold, therebydetecting said compound in a sample. In further embodiments, thescaffold is labeled (i.e., radiolabel, fluorescent, enzyme-linked orcolorimetric label) to facilitate the detection of the compound.

The invention also provides methods of capturing a compound utilizingthe Tn3 scaffolds of the invention. Based on the binding specificitiesof the Tn3 scaffolds obtained by library screening, it is possible touse such Tn3 scaffolds in assays to capture the specific target in asample, such as for purification methods. In one embodiment, the methodof capturing a compound in a sample comprises contacting said compoundin a sample with a scaffold of the invention under conditions that allowthe formation of a compound:scaffold complex and removing said complexfrom the sample, thereby capturing said compound in said sample. Infurther embodiments, the Tn3 scaffold is immobilized to facilitate theremoving of the compound:scaffold complex.

In some embodiments, Tn3 scaffolds isolated from libraries of theinvention comprise at least one, at least two, at least four, at leastfive, at least six, or more randomized loops. In some embodiments,isolated Tn3 scaffold loop sequences may be swapped from a donorscaffold to any loop in a FnIII receiver scaffold included, but notlimited to, a Tn3 receiver scaffold (for example, an FG loop sequencefrom a donor scaffold may be transferred to any loop in a receiver FnIIIscaffold). In specific embodiments, isolated loop sequences may betransferred to the cognate loop in the receiving scaffold (for example,an FG loop sequence from a donor scaffold may be transferred to an FnIIIreceiver scaffold in the FG loop position). In some embodiments,isolated loop sequences may be “mix and matched” randomly with variousreceiver scaffolds.

In other embodiments, isolated Tn3 scaffolds sequences may be identifiedby the loop sequence. For example, a library is used to pan against aparticular target and a collection of specific binders are isolated. Therandomized loop sequences may be characterized as specific sequencesindependently of the Tn3 scaffold background (i.e., the scaffold thatbinds target X wherein said scaffold comprises an FG loop sequence ofSEQ ID NO:X). In alternative embodiments, where a scaffold exhibits twoloop sequences that bind target X, the loop sequences may becharacterized as binding target X in the absence of the scaffoldsequence. In other words, it is contemplated that scaffolds isolatedfrom a library that bind a particular target may be expressed as thevariable loop sequences that bind that target independent of thescaffold backbone. This process would be analogous to the concept ofCDRs in variable regions of antibodies.

Affinity Maturation

The development of Tn3 scaffolds of the invention may involve one ormore in vitro or in vivo affinity maturation steps. In some embodiments,Tn3 monomer subunits can undergo a single step of affinity maturation.In other embodiments, Tn3 monomer subunits can under two or more stepsof affinity maturation. Any affinity maturation approach can be employedthat results, in general, in amino acid changes in a parent Tn3scaffold, or specifically amino acid changes in a parent Tn3 scaffold'sloops that improve the binding of the affinity matured Tn3 scaffold tothe desired antigen.

These amino acid changes can, for example, be achieved via randommutagenesis, “walk though” mutagenesis, and “look through” mutagenesis.Such mutagenesis can be achieved by using, for example, error-prone PCR,“mutator” strains of yeast or bacteria, incorporation of random ordefined nucleic acid changes during ab initio synthesis of all or partof a FnIII-based binding molecule. Methods for performing affinitymaturation and/or mutagenesis are described, for example, in U.S. Pat.Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479; 5,922,545; 5,830,721;5,605,793, 5,830,650; 6,194,550; 6,699,658; 7,063,943; 5,866,344 and PCTPublication WO06023144.

Such affinity maturation methods may further require that the stringencyof the antigen-binding screening assay is increased to select for Tn3scaffolds with improved affinity for an antigen. Art recognized methodsfor increasing the stringency of a protein-protein interaction assay canbe used here. In one embodiment, one or more of the assay conditions arevaried (for example, the salt concentration of the assay buffer) toreduce the affinity of the Tn3 scaffold for the desired antigen. Inanother embodiment, the length of time permitted for the Tn3 scaffold tobind to the desired antigen is reduced.

In another embodiment, a competitive binding step can be added to theprotein-protein interaction assay. For example, the Tn3 scaffold can befirst allowed to bind to a desired immobilized antigen. A specificconcentration of non-immobilized antigen is then added which serves tocompete for binding with the immobilized antigen such that the Tn3scaffolds with the lowest affinity for antigen are eluted from theimmobilized antigen resulting in selection of Tn3 scaffolds withimproved antigen binding affinity. The stringency of the assayconditions can be further increased by increasing the concentration ofnon-immobilized antigen is added to the assay.

Screening methods may also require multiple rounds of selection toenrich for one or more Tn3 scaffolds with improved antigen binding. Inone embodiment, at each round of selection further amino acid mutationsare introduce into the Tn3 scaffold. In another embodiment, at eachround of selection the stringency of binding to the desired antigen isincreased to select for Tn3 scaffolds with increased affinity forantigen.

In some embodiments, affinity maturation is performed by saturationmutagenesis of portions of the BC, DE, and FG loops of Tn3. In someembodiments, saturation mutagenesis is performed using Kunkelmutagenesis. In other embodiments, saturation mutagenesis is performedby using PCR.

In some embodiments, at least one, at least two, at least three, atleast four, at least five, or more than five rounds of affinitymaturation are applied. In some embodiments, saturation mutagenesis isapplied to only one loop, whereas in some other embodiments, only oneloop or a portion of a loop is mutated during one round of affinitymaturation. In some embodiments, more than one loop or portions of oneor more than loop are mutated during the same round of affinitymaturation.

In other embodiments, the BC, DE, and FG loops mutated simultaneouslyduring the same round of affinity maturation.

In the case of the monomers to assemble into multimeric Tn3 scaffoldsbinding to different epitopes of the same target, each bindingspecificity can be screened independently.

In some embodiments, the loops are randomized using a phage displaylibrary. In some embodiments, the binding of a Tn3 scaffold to a desiredtarget can be determined using methods recognized in the art. Also, theamino acid sequences of the Tn3 scaffolds identified in the screens canbe determined using art recognized methods.

In some embodiments, the monomeric affinity matured scaffolds of theinvention exhibit an increased in affinity for CD40L of at least 5-fold,at least 10-fold, at least 20-fold, at least 40-fold, at least 6o-fold,at least 80-fold, or at least 100-fold or more compared to the same Tn3scaffold prior to affinity maturation, as measured by Surface PlasmonResonance or by other assays known in the art. In some embodiments, themonomeric affinity matured scaffolds of the invention have adissociation constant (K_(d)) of less than 5 μM, less than 1 μM, lessthan 500 μM, less than 250 μM, less than 100 μM, or less than 50 μM, asmeasured by Surface Plasmon Resonance or by other assays known in theart.

These affinity maturation methods can be applied to develop Tn3scaffolds with desirable improved binding properties such as increasedaffinity or other desirable characteristics, such as favorablepharmacokinetic properties, high potency, low immunogenicity, increasedor decreased cross-reactivity, etc.

Generation of Tandem Repeats

Linking of tandem constructs, a dimer formed by linking twoCD40L-specific monomer subunits, may be generated by ligation ofoligonucleotides at restriction sites using restriction enzymes known inthe art, including but not limited to type II and type IIS restrictionenzymes.

The multimeric Tn3 scaffolds of the invention may comprise a linker atthe C-terminus and/or the N-terminus and/or between domains as describedherein. Further, scaffolds of the invention comprising at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8 or polypeptide scaffolds may be fused or conjugated to adimerization domain, including but not limited to an antibody moietyselected from:

-   -   (i) a Fab fragment, having VL, CL, VH and CH1 domains;    -   (ii) a Fab′ fragment, which is a Fab fragment having one or more        cysteine residues at the C-terminus of the CH1 domain;    -   (iii) a Fd fragment having VH and CH1 domains;    -   (iv) a Fd′ fragment having VH and CH1 domains and one or more        cysteine residues at the C-terminus of the CH1 domain;    -   (v) a Fv fragment having the VL and VH domains of a single arm        of an antibody;    -   (vi) a dAb fragment which consists of a VH domain;    -   (vii) isolated CDR regions;    -   (viii) F(ab′)2 fragments, a bivalent fragment including two Fab′        fragments linked by a disulphide bridge at the hinge region;    -   (ix) single chain antibody molecules (e.g., single chain Fv;        scFv);    -   (x) a “diabody” with two antigen binding sites, comprising a        heavy chain variable domain (VH) connected to a light chain        variable domain (VL) in the same polypeptide chain;    -   (xi) a “linear antibody” comprising a pair of tandem Ed segments        (VH-CH1-VH-CH1) which, together with complementary light chain        polypeptides, form a pair of antigen binding regions;    -   (xii) a full length antibody; and    -   (xiii) an Fc region comprising CH2-CH3, which may further        comprise all or a portion of a hinge region and/or a CH1 region.

Tn3 Scaffold Production

Recombinant expression of a Tn3 scaffold of the invention requiresconstruction of an expression vector containing a polynucleotide thatencodes the Tn3 scaffold. Once a polynucleotide encoding a Tn3 scaffoldhas been obtained, the vector for the production of the Tn3 scaffold maybe produced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing a Tn3 scaffold encoding nucleotide sequenceare described herein. Methods that are well known to those skilled inthe art can be used to construct expression vectors containing scaffoldpolypeptide coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding a Tn3 scaffold of theinvention, operably linked to a promoter.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce a Tn3 scaffold of the invention. Thus, theinvention includes host cells containing a polynucleotide encoding ascaffold of the invention, operably linked to a heterologous promoter.Suitable host cells include, but are not limited to, microorganisms suchas bacteria (e.g., E. coli and B. subtilis).

A variety of host-expression vector systems may be utilized to expressthe Tn3 scaffolds of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express a scaffold of the invention in situ. These includebut are not limited to microorganisms such as bacteria (e.g., E. coliand B. subtilis) transformed with recombinant bacteriophage DNA, plasmidDNA or cosmid DNA expression vectors containing scaffold codingsequences or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and3T3 cells).

Methods useful for the production of the Tn3 scaffolds of the inventionare disclosed, for example, in International Patent ApplicationPublication No WO 2009/058379. Once a scaffold of the invention has beenproduced by recombinant expression, it may be purified by any methodknown in the art for purification of a protein.

In some embodiments, scaffolds of the invention can be produced in anaglycosylated form by replacing amino acid residues that can beglycosylated during recombinant expression. In one specific embodiment,serine amino acids in a glycine-serine linker (e.g., SEQ ID NO: 131 orSEQ ID NO: 132) can be replaced by other amino acids residues such asalanine, glycine, leucine, isoleucine or valine (see, e.g., SEQ ID NOs:140, 141, 142 and 143) in order to prevent glycosylation duringrecombinant expression. In some specific embodiments, an N-glycosylationsite is removed from a Tn3 scaffolds of the invention. In otherembodiments, a scaffold of the invention can be deglycosylated afterrecombinant expression. Methods of in vitro deglycosylation afterrecombinant expression using, e.g., enzymatic cocktails are known in theart (for example, the PFGase F, Enodo F Multi, Orela O-linked GlycanRelease, Enzymatic CarboRelease, and Enzymatic DeGlycoMx deglycosylationkits marketed by QA-bio, Palm Desert, Calif.).

Production of the Tn3 scaffolds of the invention in the researchlaboratory can be scaled up to produce scaffolds in analytical scalereactors or production scale reactors, as described in U.S. PatentPublication No. US 2010-0298541 A1.

Scalable Production of Secreted Tn3 Scaffolds

The Tn3 scaffolds of the invention can be produced intracellularly or asa secreted form. In some embodiments, the secreted scaffolds areproperly folded and fully functional. Tn3 scaffolds of the invention canbe produced by a scalable process. In some embodiments, scaffolds can beproduced by a scalable process of the invention in the researchlaboratory that can be scaled up to produce the scaffolds of theinvention in analytical scale bioreactors (for example, but not limitedto 5 L, 10 L, 15 L, 30 L, or 50 L bioreactors). In other embodiments,the Tn3 scaffolds can be produced by a scalable process of the inventionin the research laboratory that can be scaled up to produce the Tn3scaffolds of the invention in production scale bioreactors (for example,but not limited to 75 L, 100 L, 150 L, 300 L, or 500 L). In someembodiments, the scalable process of the invention results in little orno reduction in production efficiency as compared to the productionprocess performed in the research laboratory.

Linkers

The monomer subunits in a multimeric Tn3 scaffold can be connected byprotein and/or nonprotein linkers, wherein each linker is fused to atleast two monomer subunits. A suitable linker can consist of a proteinlinker, a nonprotein linker, and combinations thereof. Combinations oflinkers can be homomeric or heteromeric. In some embodiments, amultimeric Tn3 scaffold of the invention comprises a plurality ofmonomer subunits wherein are all the linkers are identical. In otherembodiments, a multimeric Tn3 scaffold comprises a plurality of monomersubunits wherein at least one of the linkers is functionally orstructurally different from the rest of the linkers. In someembodiments, linkers can themselves contribute to the activity of amultimeric Tn3 scaffold by participating directly or indirectly in thebinding to a target.

In some embodiments, the protein linker is a polypeptide. The linkerpolypeptide should have a length, which is adequate to link two or moremonomer subunits in such a way that they assume the correct conformationrelative to one another so that they retain the desired activity.

In one embodiment, the polypeptide linker comprises 1 to about 1000amino acids residues, 1 to about 50 amino acid residues, 1-25 amino acidresidues, 1-20 amino acid residues, 1-15 amino acid residues, 1-10 aminoacid residues, 1-5 amino acid residues, 1-3 amino acid residues. Theinvention further provides nucleic acids, such as DNA, RNA, orcombinations of both, encoding the polypeptide linker sequence. Theamino acid residues selected for inclusion in the polypeptide linkershould exhibit properties that do not interfere significantly with theactivity or function of the multimeric Tn3 scaffold of the invention.Thus, a polypeptide linker should on the whole not exhibit a chargewhich would be inconsistent with the activity or function of the Tn3multimeric scaffold of the invention, or interfere with internalfolding, or form bonds or other interactions with amino acid residues inone or more of the monomer subunits which would seriously impede thebinding of the multimeric Tn3 scaffold of the invention to CD40L.

The use of naturally occurring as well as artificial peptide linkers toconnect polypeptides into novel linked fusion polypeptides is well knownin the literature. Accordingly, the linkers fusing two or more monomersubunits are natural linkers, artificial linkers, or combinationsthereof. In some embodiments, the amino acid sequences of all peptidelinkers present in a Tn3 multimeric scaffold of the invention areidentical. In other embodiments, the amino acid sequences of at leasttwo of the peptide linkers present in a multimeric Tn3 scaffold of theinvention are different.

In some embodiments, a polypeptide linker possesses conformationalflexibility. In some embodiments, a polypeptide linker sequencecomprises a (G-G-G-G-X)_(m) amino acid sequence where X is Alanine (A),Serine (S), Glycine (G), Isoleucine (I), Leucine (L) or Valine (V) and mis a positive integer (see, e.g., SEQ ID NO: 209). In a specificembodiment, a polypeptide linker sequence comprises a (G-G-G-G-S)_(m)amino acid sequence where m is a positive integer (see, e.g., SEQ ID NO:147). In another specific embodiment, a polypeptide linker sequencecomprises a (G-G-G-G-G)_(m) amino acid sequence where m is a positiveinteger (see, e.g., SEQ ID NO: 148). In still another specificembodiment, a polypeptide linker sequence comprises a (G-G-G-G-A)_(m)amino acid sequence where m is a positive integer (see, e.g., SEQ ID NO:149). In some embodiments, a polypeptide linker is an inherentlyunstructured natural or artificial polypeptide (see, e.g.,Schellenberger et al., Nature Biotechnol. 27:1186-1190, 2009; see also,Sickmeier et al., Nucleic Acids Res. 35:D786-93, 2007).

The peptide linker can be modified in such a way that an amino acidresidue comprising an attachment group for a non-polypeptide moiety isintroduced. Examples of such amino acid residues may be a cysteineresidue (to which the non-polypeptide moiety is then subsequentlyattached) or the amino acid sequence may include an in vivoN-glycosylation site (thereby attaching a sugar moiety (in vivo) to thepeptide linker).

In some embodiments, the amino acid sequences of all peptide linkerspresent in the polypeptide multimer are identical. Alternatively, theamino acid sequences of all peptide linkers present in the polypeptidemultimer may be different.

Labeling or Conjugation of Tn3 Scaffolds

The Tn3 scaffolds of the invention can be used in non-conjugated form orconjugated to at least one of a variety of heterologous moieties tofacilitate target detection or for imaging or therapy. The Tn3 scaffoldsof the can be labeled or conjugated either before or after purification,when purification is performed.

Many heterologous moieties lack suitable functional groups to which Tn3scaffolds of the invention can be linked. Thus, in some embodiments, theeffector molecule is attached to the scaffold through a linker, whereinthe linker contains reactive groups for conjugation. In someembodiments, the heterologous moiety conjugated to a Tn3 scaffold of theinvention can function as a linker. In other embodiments, the moiety isconjugated to the Tn3 scaffold via a linker that can be cleavable ornon-cleavable. In one embodiment, the cleavable linking molecule is aredox cleavable linking molecule, such that the linking molecule iscleavable in environments with a lower redox potential, such as thecytoplasm and other regions with higher concentrations of molecules withfree sulfhydryl groups. Examples of linking molecules that may becleaved due to a change in redox potential include those containingdisulfides.

In some embodiments, Tn3 scaffolds of the invention are engineered toprovide reactive groups for conjugation. In such scaffolds, theN-terminus and/or C-terminus can also serve to provide reactive groupsfor conjugation. In other embodiments, the N-terminus can be conjugatedto one moiety (such as, but not limited to PEG) while the C-terminus isconjugated to another moiety (such as, but not limited to biotin), orvice versa.

The term “polyethylene glycol” or “PEG” means a polyethylene glycolcompound or a derivative thereof, with or without coupling agents,coupling or activating moieties (e.g., with thiol, triflate, tresylate,aziridine, oxirane, N-hydroxysuccinimide or a maleimide moiety). Theterm “PEG” is intended to indicate polyethylene glycol of a molecularweight between 500 and 150,000 Da, including analogues thereof, whereinfor instance the terminal OH-group has been replaced by a methoxy group(referred to as mPEG).

The Tn3 scaffolds of the invention can be derivatized with polyethyleneglycol (PEG). PEG is a linear, water-soluble polymer of ethylene oxiderepeating units with two terminal hydroxyl groups. PEGs are classifiedby their molecular weights which typically range from about 500 daltonsto about 40,000 daltons. In a specific embodiment, the PEGs employedhave molecular weights ranging from 5,000 daltons to about 20,000daltons. PEGs coupled to the scaffolds of the invention can be eitherbranched or unbranched. See, for example, Monfardini, C. et al. 1995Bioconjugate Chem 6:62-69. PEGs are commercially available from NektarInc., Sigma Chemical Co. and other companies. Such PEGs include, but arenot limited to, monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS),monomethoxypolyethylene glycol-amine (MePEG-NH2),monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

Briefly, the hydrophilic polymer which is employed, for example, PEG, iscapped at one end by an unreactive group such as a methoxy or ethoxygroup. Thereafter, the polymer is activated at the other end by reactionwith a suitable activating agent, such as cyanuric halides (for example,cyanuric chloride, bromide or fluoride), carbonyldiimidazole, ananhydride reagent (for example, a dihalo succinic anhydride, such asdibromosuccinic anhydride), acyl azide, p-diazoniumbenzyl ether,3-(p-diazoniumphenoxy)-2-hydroxypropylether) and the like. The activatedpolymer is then reacted with a polypeptide as described herein toproduce a polypeptide derivatized with a polymer. Alternatively, afunctional group in the Tn3 scaffolds of the invention can be activatedfor reaction with the polymer, or the two groups can be joined in aconcerted coupling reaction using known coupling methods. It will bereadily appreciated that the polypeptides of the invention can bederivatized with PEG using a myriad of other reaction schemes known toand used by those of skill in the art. A PEG can be coupled to ascaffold of the invention at one or more functional groups at either endof the Tn3 scaffold or within the Tn3 scaffold. In certain embodiments,the PEG is coupled at either the N-terminus or the C-terminus.

In other embodiments, Tn3 scaffolds of the invention, analogs orderivatives thereof may be conjugated to a diagnostic or detectableagent. Such Tn3 scaffolds can be useful for monitoring or prognosing thedevelopment or progression of a disease as part of a clinical testingprocedure, such as determining the efficacy of a particular therapy.

The present invention further encompasses uses of Tn3 scaffoldsconjugated to a therapeutic moiety. A Tn3 scaffold may be conjugated toa therapeutic moiety such as a cytotoxin, e.g., a cytostatic orcytocidal agent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters. A cytotoxin or cytotoxic agent includes any agent thatis detrimental to cells.

Assaying Tn3 Scaffolds

The binding affinity and other binding properties of a Tn3 scaffold toan antigen may be determined by a variety of in vitro assay methodsknown in the art including for example, equilibrium methods (e.g.,enzyme-linked immunoabsorbent assay (ELISA) or kinetics (e.g., BIACORE®analysis), and other methods such as indirect binding assays,competitive binding assays, gel electrophoresis and chromatography(e.g., gel filtration). These and other methods may utilize a label onone or more of the components being examined and/or employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999).

In some embodiments, Tn3 scaffolds of the invention specifically bind atarget with specific kinetics. In some embodiments, Tn3 scaffolds of theinvention may have a dissociation constant or K_(d) (k_(off)/k_(on)) ofless than 1×10⁻²M, 1×10⁻³M, 1×10⁻⁴M, 1×10⁻⁵M, 1×10⁻⁶M, 1×10⁻⁷M, 1×10⁻⁸M,1×10⁻⁹M, 1×10⁻¹⁰M, 1×10⁻¹¹M, 1×10⁻¹²M, 1×10⁻¹³M, 1×10⁻¹⁴M or less than1×10⁻¹⁵M. In specific embodiments, Tn3 scaffolds of the invention have aK_(d) of 500 μM, 100 μM, 500 nM, 100 nM, 1 nM, 500 pM, 100 pM or less asdetermined by a BIAcore Assay® or by other assays known in the art.

In an alternative embodiment, the affinity of the Tn3 scaffolds of theinvention is described in terms of the association constant (K_(a)),which is calculated as the ratio k_(on)/k_(off), of at least 1×10²M⁻¹,1×10³M⁻¹, 1×10⁴M⁻¹, 1×10⁵M⁻¹, 1×10⁶M⁻¹, 1×10⁷M⁻¹, 1×10⁸M⁻¹, 1×10⁹M⁻¹,1×10¹⁰M⁻¹ 1×10¹¹M⁻¹ 1×10¹²M⁻¹, 1×10¹³M⁻¹, 1×10¹⁴M⁻¹, 1×10¹⁵M⁻¹, or atleast 5×10¹⁵ M⁻¹.

In certain embodiments the rate at which the Tn3 scaffolds of theinvention dissociate from a target epitope may be more relevant than thevalue of the K_(d) or the K_(a). In some embodiments, the Tn3 scaffoldsof the invention have a k_(off) of less than 10⁻³ s⁻¹, less than 5×10⁻³s⁻¹, less than 10⁻⁴ s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, lessthan 5×10⁻⁵ s⁻¹, less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than10⁻⁷ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸s⁻¹, less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In certain other embodiments, the rate at which the Tn3 scaffolds of theinvention associate with a target epitope may be more relevant than thevalue of the K_(d) or the K_(a). In this instance, the Tn3 scaffolds ofthe invention bind to a target with a k_(on) rate of at least 10⁵M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s⁻¹, at least 10⁶ M⁻¹s⁻¹, at least 5×10⁶M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least 5×10⁷ M⁻¹s⁻¹, or at least 10⁸M⁻¹s⁻¹, or at least 10⁹ M⁻¹s⁻¹.

Tn3 scaffolds of the invention may also be attached to solid supports,which are particularly useful for immunoassays or purification of thetarget antigen. Such solid supports include, but are not limited to,glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene.

CD40L-Specific Tn3 Scaffolds

The invention provides Tn3 scaffolds that specifically bind to CD40L. Inspecific embodiments, scaffolds of the invention specifically bind tohuman CD40L. In other specific embodiments, Tn3 scaffolds of theinvention bind to CD40L homologs from mouse, chicken, Rhesus,cynomolgus, rat, or rabbit. In some embodiments, Tn3 scaffolds of theinvention bind to an exposed epitope of CD40L. Such embodiments includeCD40L endogenously expressed on cells and/or cells transfected toectopically express the receptor.

In some embodiments, Tn3 scaffolds of the invention recognize epitopesdisplayed on a monomeric CD40L. In other embodiments, Tn3 scaffolds ofthe invention recognize epitopes displayed on a trimeric form of CD40L.In other embodiments, Tn3 scaffolds of the invention recognize epitopesdisplayed on a membrane bound CD40L. In other embodiments, Tn3 scaffoldsof the invention recognize epitopes displayed on soluble CD40L.

In yet other embodiments, Tn3 scaffolds of the invention bind monomericCD40L and prevent or interfere with oligomerization of CD40L molecules.In yet other embodiments, scaffolds of the invention reduce or inhibitinteraction of CD40L with CD40. In other embodiments, Tn3 scaffolds ofthe invention agonize cellular signaling mediated by CD40L. In yet otherembodiments, Tn3 scaffolds of the invention antagonize cellularsignaling mediated by CD40L.

The invention also provides methods of modulating CD40L activity usingthe Tn3 scaffolds described herein. In some embodiments, methods of theinvention comprise contacting a CD40L with CD40L-specific scaffolds andblocking the interaction between CD40 and CD40L. In other embodiments,methods of the invention comprise contacting a cell expressing CD40Lwith a CD40L-specific Tn3 scaffold and preventing proteolytic cleavageof CD40L from the cell surface. In other embodiments, methods of theinvention comprise contacting a CD40L monomer with a CD40L-specific Tn3scaffold and preventing CD40L oligomerization. In other embodiments,dimerization or oligomerization of CD40L may be achieved through the useof multimeric Tn3 scaffolds.

In some embodiments, methods of the invention comprise theadministration of a CD40L specific scaffold that reduces a CD40-mediatedimmune response (see, e.g., Elqueta et al. 229: 152-172, 2009), or adownstream signaling pathway initiated by CD40 binding to CD40L, asmeasured by routine assays known in the art.

Without wishing to be bound by any particular theory, CD40L scaffolds ofthe present invention could function by preventing binding of CD40L toCD40, by binding and sequestering soluble CD40L, by altering theinteraction of CD40L with CD40 but not preventing binding, by preventingor enhancing metalloprotease-mediated enzymatic cleavage of CD40L fromthe cell surface to yield soluble CD40L, by preventing or enhancing cellsurface CD40L endocytosis, etc.

Specific CD40L Binding Sequences

In some embodiments, the Tn3 scaffold of the invention compriseCD40L-specific monomer subunits comprising at least one, at least two,at least three, at least four, at least five, or at least six loopsequences that bind to CD40L.

In some embodiments, CD40L-specific monomer subunits comprise at leastone, at least two, at least three, at least four, at least five, or atleast six loop sequences of CD40L-binding monomer clones selected from:309 (parental 309 family clone isolated from naive Tn3 library; SEQ IDNO: 20), 309FGwt (parental 309 clone with humanized FG loop; SEQ ID NO:22), 340 (affinity matured 309 clone; SEQ ID NO: 24), 341 (affinitymatured 309 clone; SEQ ID NO: 26), 342 (affinity matured 309 clone; SEQID NO: 28 or SEQ ID NO: 146), 343 (affinity matured 309 clone; SEQ IDNO: 30), 344 (affinity matured 309 clone; SEQ ID NO: 32), 345 (affinitymatured 309 clone; SEQ ID NO: 34), 346 (affinity matured 309 clone; SEQID NO: 36), 347 (affinity matured 309 clone; SEQ ID NO: 38), 348(affinity matured 309 clone; SEQ ID NO: 40), 349 (affinity matured 309clone; SEQ ID NO: 42), 311 (parental 311 family clone isolated fromnaive Tn3 library; SEQ ID NO: 44), 311K4E (variant 311 family clone fromfirst round of affinity maturation; SEQ ID NO: 46); 311K4E_1 (variant311 family clone from second round of affinity maturation; SEQ ID NO:48), 311K4E_2 (variant 311 family clone from second round of affinitymaturation; SEQ ID NO: 50), 311K4E_3 (variant 311 family clone fromsecond round of affinity maturation; SEQ ID NO: 52), 311K4E_4 (variant311 family clone from second round of affinity maturation; SEQ ID NO:54), 311K4E_5 (variant 311 family clone from second round of affinitymaturation; SEQ ID NO: 56), 311K4E_7 (variant 311 family clone fromsecond round of affinity maturation; SEQ ID NO: 58), 311K4E_8 (variant311 family clone from second round of affinity maturation; SEQ ID NO:60), 311K4E_9 (variant 311 family clone from second round of affinitymaturation; SEQ ID NO: 62), 311K4E_10 (variant 311 family clone fromsecond round of affinity maturation; SEQ ID NO: 64), 311K4E_11 (variant311 family clone from second round of affinity maturation; SEQ ID NO:66), 311K4E_12 (variant 311 family clone from second round of affinitymaturation; SEQ ID NO: 68), 311K4E_13 (variant 311 family clone fromsecond round of affinity maturation; SEQ ID NO: 70), 311K4E_14 (variant311 family clone from second round of affinity maturation; SEQ ID NO:72), 311K4E_15 (variant 311 family clone from second round of affinitymaturation; SEQ ID NO: 74), 311K4E_16 (variant 311 family clone fromsecond round of affinity maturation; SEQ ID NO: 76), 311K4E_19 (variant311 family clone from second round of affinity maturation; SEQ ID NO:78), 311K4E_20 (variant 311 family clone from second round of affinitymaturation; SEQ ID NO: 80), and 311K4E_21 (variant 311 family clone fromsecond round of affinity maturation; SEQ ID NO: 82).

In some embodiments, CD40L-specific monomer subunits comprise at leastone loop sequence selected from the loop sequences listed in TABLE 2. Inother embodiments, CD40L-specific monomer subunits comprise at least oneBC loop sequence selected from the BC loop sequences listed in TABLE 2.In other embodiments, CD40L-specific monomer subunits comprise at leastone DE loop sequence selected from the DE loop sequences listed in TABLE2. In other embodiments, CD40L-specific monomer subunits comprise atleast one FG loop sequence selected from the FG loop sequences listed inTABLE 2.

In some embodiments, CD40L-specific monomer subunits comprise a BC loopsequence selected from the BC loop sequences listed in TABLE 2; and a DEloop sequence selected from the DE loop sequences listed in TABLE 2. Inother embodiments, CD40L-specific monomer subunits comprise a BC loopsequence selected from the BC loop sequences listed in TABLE 2; and anFG loop sequence selected from the FG loop sequences listed in TABLE 2.In other embodiments, CD40L-specific monomer subunits comprise a DE loopsequence selected from the DE loop sequences listed in TABLE 2; and anFG loop sequence selected from the FG loop sequences listed in TABLE 2.In some embodiments, a CD40L-specific monomer subunits comprises loopsequences corresponding to loop sequences from one, two or threedifferent Tn3 clones.

In certain embodiments, where the CD40L-specific monomer scaffoldsequence contains a linker and/or a Histidine tag (e.g., a His-8 tag) atthe C-terminus of the sequence, or additional N-terminal amino acids,these C-terminal linker and/or Histidine tag and additional N-terminalamino acids can be removed, the corresponding amino acid sequence thuscontaining a deletion of the C-terminal linker and His tag sequences andthe N-terminal additional amino acid or amino acids.

In some embodiments, the CD40L-specific Tn3 scaffold comprises a singlemonomer subunit, e.g., the 342 clone sequence (affinity matured 309clone; SEQ ID NO: 28 and/or SEQ ID NO: 146). In other embodiments, theCD40L-specific scaffold comprises more than one monomer subunits, e.g.,two 342 clone monomer subunits (SEQ ID NO: 28 and/or SEQ ID NO: 146) intandem (see, e.g., SEQ ID NO: 135). In specific embodiments, Tn3scaffolds of the invention are conjugated to a variant HSA (see, e.g.,SEQ ID NO: 134 and SEQ ID NO: 135). In further embodiments, the HSA canbe conjugated at either the N-terminus or the C-terminus of themultimeric Tn3 scaffold.

In a specific embodiment, the CD40L-specific Tn3 scaffold comprises asingle 311K4E_12 monomer subunit, a GS linker, and a C34S HSA variant(see, e.g., SEQ ID NO: 201). In another specific embodiment, theCD40L-specific Tn3 scaffold comprises a single 311K4E_12 monomer subunitwith a beta strand C CELTYG variant, an all glycine linker, and a C34SHSA variant (see, e.g., SEQ ID NO: 202). In another specific embodiment,the CD40L-specific Tn3 scaffold comprises two 311K4E_12 subunits intandem, and two GS linkers, wherein one GS linker connects the subunitsto each other and a second GS linker connects one subunit to a C34S HSAvariant (see, e.g., SEQ ID NO: 203). In yet another specific embodiment,the CD40L-specific Tn3 scaffold comprises two 311K4E_12 subunits intandem, and two all glycine linkers, wherein one all glycine linkerconnects the subunits to each other and a second all glycine linkerconnects one subunit to a C34S HSA variant (see, e.g., SEQ ID NO: 204).

In one specific embodiment, the CD40L-specific Tn3 scaffold comprisestwo 309 subunits connected in tandem via a GS linker (see, e.g., SEQ IDNO: 205). In another specific embodiment, the CD40L-specific Tn3scaffold comprises a single 309 subunit connected to a C34S HSA variant(see, e.g., SEQ ID NO: 206). In another specific embodiment, theCD40L-specific Tn3 scaffold comprises two 309 subunits in tandem, andtwo GS linkers, wherein one GS linker connects the subunits to eachother and a second GS linker connects one subunit to a C34S HSA variant(see, e.g., SEQ ID NO: 207).

In a specific embodiment, the CD40L-specific Tn3 scaffold comprises asingle 342 monomer subunit, a GS linker, and a C34S HSA variant (see,e.g., SEQ ID NO: 134). In another specific embodiment, theCD40L-specific Tn3 scaffold comprises a single 342 monomer subunit, anall glycine linker, and a C34S HSA variant (see, e.g., SEQ ID NO: 144).In another specific embodiment, the CD40L-specific Tn3 scaffoldcomprises two 342 subunits in tandem, and two GS linkers, wherein one GSlinker connects the subunits to each other and a second GS linkerconnects one subunit to a C34S HSA variant (see, e.g., SEQ ID NO: 135).In yet another specific embodiment, the CD40L-specific Tn3 scaffoldcomprises two 342 subunits in tandem, and two all glycine linkers,wherein one all glycine linker connects the subunits to each other and asecond all glycine linker connects one subunit to a C34S HSA variant(see, e.g., SEQ ID NO: 145). In yet another specific embodiment, theCD40L-specific Tn3 scaffold comprises two 342 subunits connected intandem by a GS linker (see, e.g., SEQ ID NO: 208).

In a specific embodiment, the CD40L-specific Tn3 scaffold comprises Inanother specific embodiment, the CD40L-specific Tn3 scaffold comprises a311 subunit, or a subunit derived from 311 (e.g., 311K4E_12) and a 309subunit, or a subunit derived from 309 (e.g., 342) in tandem and two GSlinkers, wherein one GS linker connects the subunits to each other and asecond GS linker connects one subunit to a C34S HSA variant (see, e.g.,SEQ ID NO: 135). In yet another specific embodiment, the CD40L-specificTn3 scaffold comprises a 311 subunit, or a subunit derived from 311(e.g., 311K4E_12) and a 309 subunit, or a subunit derived from 309(e.g., 342) in tandem, and two all glycine linkers, wherein one allglycine linker connects the subunits to each other and a second allglycine linker connects one subunit to a C34S HSA variant (see, e.g.,SEQ ID NO: 145).

Examples of CD40L-specific tandem bivalent Tn3 scaffolds and SerumAlbumin (SA) fusions are shown in FIG. 2A (also see FIG. 9A). Althoughspecific linkers are provided in FIG. 2A, other linkers are contemplatedas provided herein. Although wild type mature SA may be used, e.g.,murine serum albumin (MSA) or human serum albumin (HSA), it iscontemplated that one or more Cysteine (C) amino acid residues in themature SA may be substituted, for example with Serine (S), Alanine (A),Glycine (G), etc.

Representative constructs are shown below. The sequence of the SA isunderlined. Linkers are boxed. It will be understood that numerousvariations are within the scope of the invention. For example, thelinkers may be altered (several non-limited examples are providedherein), the first one or two N-terminal amino acid residues (SQ) may beabsent and/or substituted with alternative amino acid residues, a tag(e.g., 6×His tag) may be incorporated, alternative CD40L-specificscaffolds (e.g., those based on the 10^(th) Fn3 domain of fibronectin)may be utilized in a similar construct, etc.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, forexample, but not limited to, a pharmaceutical composition, containingone or a combination of Tn3 scaffolds of the present invention,formulated together with a pharmaceutically acceptable carrier. Suchcompositions may include one or a combination of, for example, but notlimited to two or more different Tn3 scaffolds of the invention. Forexample, a pharmaceutical composition of the invention may comprise acombination of Tn3 scaffolds that bind to different epitopes on thetarget antigen or that have complementary activities. In a specificembodiment, a pharmaceutical composition comprises a single monomer Tn3scaffold of the invention. In a specific embodiment, a pharmaceuticalcomposition comprises a multimeric Tn3 scaffold of the invention. Instill another specific embodiment, a pharmaceutical compositioncomprises dimer Tn3 scaffold of the invention.

Pharmaceutical compositions of the invention also can be administered incombination therapy, such as, combined with other agents. For example,the combination therapy can include a Tn3 scaffold of the presentinvention combined with at least one other therapy wherein the therapymay be immunotherapy, chemotherapy, radiation treatment, or drugtherapy. The pharmaceutical compounds of the invention may include oneor more pharmaceutically acceptable salts.

Methods of Using Scaffolds

The Tn3 scaffolds of the present invention have in vitro and in vivodiagnostic and therapeutic utilities. For example, these molecules canbe administered to cells in culture, e.g., in vitro or ex vivo, or in asubject, e.g., in vivo, to treat, prevent or diagnose a variety ofdisorders.

The invention also provides methods of using the Tn3 scaffolds of theinvention. The present invention also encompasses the use of the Tn3scaffolds of the invention for the prevention, diagnosis, management,treatment or amelioration of one or more symptoms associated withdiseases, disorders of diseases or disorders, including but not limitedto cancer, inflammatory and autoimmune diseases, infectious diseaseseither alone or in combination with other therapies. The invention alsoencompasses the use of the Tn3 scaffolds of the invention conjugated orfused to a moiety (e.g., therapeutic agent or drug) for prevention,management, treatment or amelioration of one or more symptoms associatedwith diseases, disorders or infections, including but not limited tocancer, inflammatory and autoimmune diseases, infectious diseases eitheralone or in combination with other therapies.

The invention also provides methods of targeting epitopes not easilyaccomplished with traditional antibodies. For example, in oneembodiment, the Tn3 scaffolds the invention may be used to first targetan adjacent antigen and while binding, another binding domain may engagethe cryptic antigen.

The invention also provides methods of using the Tn3 scaffolds to bringtogether distinct cell types. In one embodiment, the proteins of theinvention may bind a target cell with one binding domain and recruitanother cell via another binding domain. In another embodiment, thefirst cell may be a cancer cell and the second cell is an immuneeffector cell such as an NK cell. In another embodiment, the Tn3scaffolds of the invention may be used to strengthen the interactionbetween two distinct cells, such as an antigen presenting cell and a Tcell to possibly boost the immune response.

The invention also provides methods of using the Tn3 scaffolds todeplete a cell population. In one embodiment, methods of the inventionare useful in the depletion of the following cell types: eosinophil,basophil, neutrophil, T cell, B cell, mast cell, monocytes and tumorcells.

The invention also provides methods of using Tn3 scaffolds as diagnosticreagents. Such diagnostic reagents are could be used to test for thepresence or absence of CD40L, the presence of CD40 receptor, the bindingefficiency of CD40L to CD40 receptor, free CD40L in a patient, freeCD40L in a sample, or bound CD40L to CD40 receptor in a sample.

The Tn3 scaffolds of the invention and compositions comprising the sameare useful for many purposes, for example, as therapeutics against awide range of chronic and acute diseases and disorders including, butnot limited to, autoimmune and/or inflammatory diseases. Thecompositions and methods of the invention described herein are usefulfor the prevention or treatment of autoimmune disorders and/orinflammatory disorders.

Examples of autoimmune and/or inflammatory disorders include, but arenot limited to, alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunediseases of the adrenal gland, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome,psoriasis, atherosclerosis, diabetic and other retinopathies,retrolental fibroplasia, age-related macular degeneration, neovascularglaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease),corneal and other tissue transplantation, and chronic inflammation,sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusioninjury, septicemia, endotoxic shock, cystic fibrosis, endocarditis,psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock,organ ischemia, reperfusion injury, spinal cord injury and allograftrejection. autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserythematosus, Meniere's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteritis/giant cell arteritis, ulcerative colitis,uveitis, vasculitis such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis.

Examples of inflammatory disorders include, but are not limited to,asthma, encephalitis, inflammatory bowel disease, chronic obstructivepulmonary disease (COPD), allergic disorders, septic shock, pulmonaryfibrosis, undifferentiated spondyloarthropathy, undifferentiatedarthropathy, arthritis, inflammatory osteolysis, and chronicinflammation resulting from chronic viral or bacterial infections. Thecompositions and methods of the invention can be used with one or moreconventional therapies that are used to prevent, manage or treat theabove diseases.

The invention provides methods for preventing, managing, treating orameliorating cancer, autoimmune, inflammatory or infectious diseases orone or more symptoms or one or more symptoms thereof, said methodscomprising administering to a subject in need thereof one or more Tn3scaffolds of the invention in combination with one or more oftherapeutic agents that are not cancer therapeutics (a.k.a., non-cancertherapies).

Examples of such agents include, but are not limited to, anti-emeticagents, anti-fungal agents, anti-bacterial agents, such as antibiotics,anti-inflammatory agents, and antiviral agents. Non-limiting examples ofanti-emetic agents include metopimazin and metochlopramide. Non-limitingexamples of antifungal agents include azole drugs, imidazole, triazoles,polyene, amphotericin and ryrimidine. Non-limiting examples ofanti-bacterial agents include dactinomycin, bleomycin, erythromycin,penicillin, mithramycin, cephalosporin, imipenem, axtreonam, vancomycin,cycloserine, bacitracin, chloramphenicol, clindamycin, tetracycline,streptomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin,spectinomycin, trimethoprim, norfloxacin, refampin, polymyxin,amphotericin B, nystatin, ketocanazole, isoniazid, metronidazole andpentamidine. Non-limiting examples of antiviral agents includenucleoside analogs (e.g., zidovudine, acyclivir, gangcyclivir,vidarbine, idoxuridine, trifluridine and ribavirin), foscaret,amantadine, rimantadine, saquinavir, indinavir, ritonavir, interferon(“IFN”)-α,β or γ and AZT. Non-limiting examples of anti-inflammatoryagents include non-steroidal anti-inflammatory drugs (“NSAIDs”),steroidal anti-inflammatory drugs, beta-agonists, anti-cholingenicagents and methylxanthines.

In one embodiment, the invention comprises compositions capable oftreating chronic inflammation. In one embodiment, the compositions areuseful in the targeting of immune cells for destruction or deactivation.In one embodiment, the compositions are useful in targeting activated Tcells, dormant T cells, B cells, neutrophils, eosinophils, basophils,mast cells, or dendritic cells. In another embodiment, the inventioncomprises compositions capable of decreasing immune cell function. Inanother embodiment, the compositions are capable of ablating immune cellfunction.

In another embodiment, the invention comprises compositions useful fortreatment of diseases of the gastrointestinal tract. The scaffolds ofthe invention exhibit a high level of stability under low pH conditions.The stability at low pH suggests that the composition will be suitablefor oral administration for a variety of gastrointestinal disorders,such as irritable bowel syndrome, gastroesophageal reflux, intestinalpseudo-obstructions, dumping syndrome, intractable nausea, peptic ulcer,appendicitis, ischemic colitis, ulcerative colitis, gastritis,Helicobacter pylori disease, Crohn's disease, Whipple's disease, celiacsprue, diverticulitis, diverticulosis, dysphagia, hiatus hernia,infections esophageal disorders, hiccups, rumination and others.

The invention further provides combinatorial compositions and methods ofusing such compositions in the prevention, treatment, reduction, oramelioration of disease or symptoms thereof. The Tn3 scaffolds of theinvention may be combined with conventional therapies suitable for theprevention, treatment, reduction or amelioration of disease or symptomsthereof. Exemplary conventional therapies can be found in thePhysician's Desk Reference (56th ed., 2002 and 57th ed., 2003). In someembodiments, Tn3 scaffolds of the invention may be combined withchemotherapy, radiation therapy, surgery, immunotherapy with a biologic(antibody or peptide), small molecules, or another therapy known in theart. In some embodiments, the combinatorial therapy is administeredtogether. In other embodiments, the combinatorial therapy isadministered separately.

The invention also provides methods of diagnosing diseases. The Tn3scaffolds of the invention which bind a specific target associated witha disease may be implemented in a method used to diagnose said disease.In one embodiment, the Tn3 scaffolds of the invention are used in amethod to diagnose a disease in a subject, said method comprisingobtaining a sample from the subject, contacting the target with the Tn3scaffold in said sample under conditions that allow the target:scaffoldinteraction to form, identifying the target:scaffold complex and therebydetecting the target in the sample. In other embodiments, the disease tobe diagnosed is described herein.

The invention also provides methods of imaging specific targets. In oneembodiment, Tn3 scaffolds of the invention conjugated to imaging agentssuch as green-fluorescent proteins, other fluorescent tags (Cy3, Cy5,Rhodamine and others), biotin, or radionuclides may be used in methodsto image the presence, location, or progression of a specific target. Insome embodiments, the method of imaging a target comprising a Tn3scaffold of the invention is performed in vitro. In other embodiments,the method of imaging a target comprising a Tn3 scaffold of theinvention is performed in vivo. In other embodiments, the method ofimaging a target comprising a Tn3 scaffold of the invention is performedby MRI, PET scanning, X-ray, fluorescence detection or by otherdetection methods known in the art.

The invention also provides methods of monitoring disease progression,relapse, treatment, or amelioration using the scaffolds of theinvention. In one embodiment, methods of monitoring disease progression,relapse, treatment, or amelioration is accomplished by the methods ofimaging, diagnosing, or contacting a compound/target with a Tn3 scaffoldof the invention as presented herein.

Pharmaceutical Dosing and Administration

To prepare pharmaceutical or sterile compositions including a Tn3scaffold of the invention, a scaffold is mixed with a pharmaceuticallyacceptable carrier or excipient. For administration compositions arepreferably pyrogen-free which are substantially free of endotoxinsand/or related pyrogenic substances. Selecting an administration regimenfor a therapeutic depends on several factors, including the serum ortissue turnover rate of the entity, the level of symptoms, theimmunogenicity of the entity, and the accessibility of the target cellsin the biological matrix. In certain embodiments, an administrationregimen maximizes the amount of therapeutic delivered to the patientconsistent with an acceptable level of side effects.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A composition of the present invention may also be administered via oneor more routes of administration using one or more of a variety ofmethods known in the art. In certain embodiments, the Tn3 scaffolds ofthe invention can be formulated to ensure proper distribution in vivo.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

EXAMPLES

The invention is now described with reference to the following examples.These examples are illustrative only and the invention should in no waybe construed as being limited to these examples but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1 Construction of a 3 Loop Library on the Parent Tn3 Scaffold

A library was constructed based upon the parent Tn3 scaffold, describedin International Patent Application Publ. No. WO 2009/058379, wherein itis designated “Tn3 SS4.” The library contained randomized regions of theBC, DE and FG loops. This design incorporated characterized sequence andloop length diversity into the Tn3 library, consistent with patterns ofdiversity described for natural FnIII domains, three different lengthsfor the BC and FG loops, and used a “NHT” mixed codon scheme forintroducing diversity into the library (H=A, T, C). This schemegenerated 12 codons that coded for 12/20 amino acids (see TABLE 3), thatis, each codon coded for a unique amino acid. Moreover, there were nostop or Cysteine (Cys) codons.

TABLE 3 A AAT = Asn ATT = Ile ACT = Thr G GAT = Asp GTT = Val GCT = AlaC CAT = His CTT = Leu CCT = Pro T TAT = Tyr TTT = Phe TCT = Ser A T C

The library diversity was generated using the degenerateoligonucleotides shown in TABLE 4.

TABLE 4 SEQ Oligo Loop Sequence ID NO BC9 BCACCGCGCTGATTACCTGGNHTNHTSCGNHTGSTNH 178 NHTTNHTNHTGGCTGTGAACTGACCTATGGCATTAAA BC11 BCACCGCGCTGATTACCTGGNHTNHTBSTNHTNHTNH 179 NHTTNHTNHTNHTNHTGGCTGTGAACTGACCTATGGCA TTAAA BC12 BCACCGCGCTGATTACCTGGNHTVMACCGNHTNHTNH 180 NHTTRRCRGCNHTVTTNHTGGCTGTGAACTGACCTATG GCATTAAA DE DECGATCGCACCACCATAGATCTGNHTNHTNHTNHTN 181 NHTHTNHTTATAGCATTGGTAACCTGAAACCG FG9 FG GAATATGAAGTGAGCCTGATTTGCNHTAMSNHTNH182 NHT TGGTNHTNHTNHTKCGAAAGAAACCTTTACCACCG GTG FG10 FGGAATATGAAGTGAGCCTGATTTGCNHTAMSNHTNH 183 NHTTNHTNHTRGCNHTCCGGCGAAAGAAACCTTTACCA CCGGTG FG11 FGGAATATGAAGTGAGCCTGATTTGCNHTAMSNHTNH 184 NHTTGGTNHTNHTAGCAACCCGGCGAAAGAAACCTTTA CCACCGGTG Nucleotide codes: N= G/A/T/C; H = A/T/C; R = A/G; S = G/C; B = T/C/G; V = A/C/G; M = A/C; K= G/T

The library was assembled using the oligonucleotides shown in TABLE 5.

TABLE 5 SEQ Oligo Sequence ID NO BCX-DECAGATCTATGGTGGTGCGATCGCCCGGCACATC 185 bridge v2 TTTAATGCCATAGGTCAGTTCACADE-FGX GCAAATCAGGCTCACTTCATATTCGGTATCCGG 186 bridge v2TTTCAGGTTACCAATGCTAT KpnI amp CGGGTCGGTTGGGGTACCGCCACCGGTGGTAAA 187rev v2 GGTTTCTTT KpnI CGGGTCGGTTGGGGTA 188 reverse v2 BC libraryGGCCCAGCCGGCCATGGCCGCCATTGAAGTGAA 189 amp v2AGATGTGACCGATACCACCGCGCTGATTACCTG G

A mix of the degenerate oligonucleotides (equimolar ratios of theoligonucleotides corresponding to the BC and FG loops, respectively),BCX-DE bridge v2, DE-FGX bridge v2, and KpnI amp rev v2, was assembledin a 20 cycle PCR reaction without an excess of external primers. Thisproduct was diluted and amplified in a regular PCR reaction using theprimers BC library amp v2 and KpnI reverse v2. The resulting PCR productgenerated a complete Tn3 gene which was then digested with NcoI and KpnIand ligated into the phage display vector (described in WO 2009/058379).The DNA was transformed into E. coli by electroporation. The finaldiversity of the library was estimated to be about 7.9×10¹⁰ members.

After electroporation, the library was incubated for 1 hour at 37° C.with shaking. M13K07 helper phage was added and after one hour the cellswere diluted to a larger volume and grown at 37° C. with shakingovernight. The next day phage were removed and concentrated from thesupernatant by precipitation with PEG 8000.

Example 2 Panning Libraries for Human CD40L-Specific Tn3 Scaffolds

Phage displayed Tn3 libraries containing >10¹⁰ unique sequences werepanned against CD40L. The diversity in these libraries was derived fromsequence and length variability in the BC, DE and FG loops which areanalogous to the three CDR loops within an antibody variable domain.Selection of lead Tn3 proteins was performed by panning of libraries onrecombinant biotinylated human CD40L and a CD40L overexpressing CHO cellline. Alternate rounds of panning against these two reagents were usedto ensure leads would recognize the recombinant extracellular domain aswell as native, membrane-anchored CD40L.

Recombinant human CD40L (Human MegaCD40L; Axxora) was biotinylated withEZ-Link sulfo-NHS-biotin (Pierce, Rockford, Ill.) using a 5-fold molarexcess of the biotinylation reagent. After incubation for 1 hour at roomtemperature, the sample was dialyzed in PBS overnight to removeunconjugated biotin. 10 μg biotinylated CD40L was immobilized on M280streptavidin beads (Dynal, Carlsbad, Calif.), followed by blocking inPBS containing 10 mg/ml BSA for 2 hours. Input consisted of librariesdeveloped as described in Example 1 or additionally, libraries developedusing standard construction techniques, such as described in WO2009/058379.

Phages were blocked in PBS containing 10 mg/ml BSA for 2 hours. Theblocked input was added to blocked M280 streptavidin control beads(without target) and incubated on rocker for 2 hours at room temperatureto deplete the library of binders to the beads. The depleted library wasthen added to the CD40L coated beads and incubated for 2 hours at roomtemperature on a rocking platform. After three washes with PBST(PBS+0.1% Tween) to remove unbound phage, the beads were added toexponentially growing E. coli XL-1 Blue cells, which were subsequentlyco-infected with M13KO7 helper phage in 60 ml 2×YT medium containing 50μg/ml carbenicillin. After growing overnight at 37° C. with shaking,phage were harvested by PEG precipitation from the overnight culturemedia.

The second round of panning (Round 2) was performed on a CD40Loverexpressing CHO cell line. The phage library was blocked in 3%BSA/PBS rocking at room temperature for 1 hour. Cells were detached withAccutase (Invitrogen), washed 2× with 5 ml PBS, and 10⁷ cells wereblocked in 1 ml 3% BSA/PBS rocking at room temperature for 30 minutes.The blocked cells were spun down at 500×g, 5 minutes, gently resuspendedin the blocked phage library solution, and incubated 1 hour at roomtemperature. Unbound phage was removed by gently washing the cells 3times in 1 ml 3% BSA/PBS and once in PBS, pelleting cells bycentrifuging 500×g for 5 minutes in a microcentrifuge using a freshEppendorf tube for each wash. The cell pellet was added directly toexponentially growing E. coli XL-1 Blue, which were then processed asdescribed for Round 1.

Panning Round 3 was performed as described for Round 1, except boundphages were eluted by addition of 100 mM HCl followed by neutralizationwith 1M Tris-HCl, pH 8. Eluted, neutralized phage was used to infect E.coli XL1 Blue cells as described for Round 1.

Panning Round 4 was again carried out on cells as described for Round 2,except 5 washes in 3% BSA/PBS were conducted. Round 5 was done using 5μg of biotinylated MegaCD40L, but otherwise as described for Round 3.

After 5 rounds of panning, screening of resulting Tn3 variants assoluble protein was performed. Amplified and PEG precipitated phagestocks were used in a PCR to amplify a pool of fragments encompassingthe encoded Tn3 sequences. This fragment pool was digested withNcof+KpnI and cloned into the corresponding NcoI−KpnI sites of plasmidpSec-oppA(L25M)-Tn3 (see, for example, WO 2009/058379). Auto-inducingMagicMedia (Invitrogen) containing carbenicillin (100 μg/ml) in 96 welldeep-well plates were inoculated with E. coli BL21 DE3 cells transformedwith the pSec-oppA(L25M)-Tn3 derived constructs. Cultures were grown for18 hours shaking at 37° C., and cells were separated from the media bycentrifugation. The media containing secreted, soluble Tn3 variants wasused directly in a screening assay for CD40L binding.

Ten sets of 96 clones were screened to identify Tn3 proteins thatspecifically bound to recombinant CD40L. Briefly, the screening assayutilized capture of soluble His-tagged Tn3 variants secreted into mediathrough binding to an anti-His antibody immobilized in wells ofmicroplates. After capture, media and excess protein were washed awayand the interaction between captured Tn3 variants and CD40L wasmonitored by utilizing biotinylated Human MegaCD40L and measuring theremaining target (after washing the plate) by SA-HRP and conventionalELISA reagents.

In the capture step, the immobilized anti-His antibody was saturatedwith Tn3, and the molar amount of captured Tn3 in each well becamevirtually identical irrespectively of the expression levels ofindividual clones. This normalization of Tn3 levels resulted in assaylevels proportional with the efficiency of target interaction andunaffected by potential differences in protein expression levels.

Positives from this assay were sequenced to identify 34 unique Tn3sequences that bound recombinant CD40L. From the panel of uniqueCD40L-binding Tn3 sequences, a subset of 24 clones that had a robustassay signal and good expression levels judging by SDS-PAGE of theculture supernatants underwent re-expression and small scalepurification.

Briefly, Superbroth media containing carbenicillin (100 μg/mL) with 1%glucose) was inoculated with E. coli BL21 DE3 cells transformed with thepSec-oppA(L25M)-Tn3 derived constructs. Cultures were grown at 37° C. toan optical density (O.D.) of 0.5-0.8 then induced with 0.2 mM IPTG.After shaking at 37° C. for 5 hours, cells were separated from the mediaby centrifugation. Purification of Tn3 scaffolds from the media waseffected by batch purification using Ni-NTA Superflow (Qiagen), washingin 2×PBS with 20 mM imidazole, and elution with 2×PBS with 250 mMimidazole. Samples were dialyzed in PBS, and concentrations determinedby UV absorbance at 280 nm according to Gill and von Hippel (Anal.Biochem. 182: 319, 1989).

Based on assay ranking and SEC behavior, expression of 8 leads wasscaled up and purified to low endotoxin levels (<1 EU/mg) for testing ina functional cell assay.

Two Tn3 clones (designated 309 and 311) showed similar activity inbiochemical and cell-based assays (FIGS. 6A, 6B, 6C), and were 3-5 foldmore potent than the nearest rival clone.

Human CD40L-specific monovalent Tn3 scaffolds 309 and 311 inhibitedtotal B cell number, plasma cell number and Ig class switching (FIGS.6A, 6B and 6C). FIG. 6 A shows the inhibitory effect of 309 and 311 onHuCD40L-induced CD86 expression on CD19 positive human PBMCs stimulatedwith Jurkat D1.1 cells; FIG. 6B shows inhibition of HuCD40L stimulatedB-cell proliferation by 309 and 311; and FIG. 6C C shows inhibition ofplasma cell number in T/B cell co-cultures. 309 was also shown to bindactivated primary T cells by FACS (data not shown). PBMCs werestimulated by recombinant human MegaCD40L (Axxora) or human CD40Lexpressing Jurkat cells (D1.1, ATCC), and the percentage of CD19+/CD86+cells was measured by FACS after 24 hours.

The 309 and 311 lead clones were monodispersed and did not display anytendency to aggregate or form higher order oligomers in solution asdetermined by size exclusion chromatography (SEC) analysis of purifiedsamples (FIG. 7A).

The thermal stabilities of the 309 and 311 lead clones were determinedby differential scanning calorimetry (DSC) using protein samples at 1mg/mL in PBS pH 7.2 and compared to the thermal stability of theparental Tn3 protein (FIG. 7B). The 309 and 3011 lead clones had T_(m)'sof 70±1° C. which was only slightly below the T_(m) of parent Tn3 (72°C.).

As no murine cross-reactive clones were identified a similar panningprocess as described above was carried out to identify the murinespecific Tn3 designated M13. M13 also showed activity in a PBMC cellbased assay (see FIG. 1A)

Example 3 CD40L-Specific Tn3 Scaffold Lead Optimization

Affinity optimization was used to increase the potency of the selectedTn3 leads. In general, one or more rounds of mutagenesis within the Tn3loops contacting the target were conducted, with selection of improvedvariants from combinatorial phage display libraries.

3.1 Loop Swapping

In order to determine which of the 3 loops in the two leads wereinvolved in interaction with CD40L, constructs were generated in whicheach single loop sequence was changed to the parent Tn3 loop sequence asfound in human tenascin C. Activities of the mutated variants werecompared to the original variants in a binding assay conducted asdescribed for the screening assay described above (FIG. 10A). For bothleads mutating the BC and DE loops resulted in a complete loss ofbinding to CD40L, whereas changing the FG loop to the parent Tn3sequence had either no effect (for 309) or limited effect (311) onbinding. Thus, the BC and DE loop appeared to contain the sequencesmainly responsible for contacting CD40L, and were thus primarilyselected for affinity optimization.

3.2 CD40L-Specific Tn3 Scaffold 309 Lead Optimization

As the loop swapping experiment indicated the 309 FG-loop sequence couldbe substituted with the parent Tn3 FG-loop sequence without substantialloss of binding potency, it was decided to use this construct (termed309FGwt) as a backbone for affinity maturation. This would eliminatenon-essential mutations deviating from the parent tenascin C sequence inorder to reduce possible immunogenicity risk. It should be noted thatthe parent Tn3 FG-loop sequence contained an RGD motif which was latereliminated by a mutation in the final lead molecules. Three BC looplibraries and one DE-loop library were generated.

For the three BC loop libraries three rounds of PCR were done using thedegenerated oligos BC9 PCR, BC 9-loop NNK and 309 BC-loop NNKdope (TABLE6) together with the reverse primer Kpn1 amp rev v2 (TABLE 5) using a309FGwt derived template in which the BC-loop codons had been replacedwith stop codons. Subsequently PCR amplification of those fragments withthe primers BC library amp v2 (TABLE 5) and KpnI reverse v2 gave thefull length Tn3 library fragment.

For the DE-loop library a PCR amplification with DE PCR and KpnI ampreverse v2 on a 309FGwt derived template (in which the DE-loop codonshad been replaced with stop codons) gave a fragment containing therandomized DE loop and wild type Tn3 FG-loop which was combined with afragment encoding the Tn3 region upstream of the DE loop generated byPCR with BC library amp v2 and BCX-DE bridge v2 on a 309 template. Thetwo fragments were joined in an overlap PCR with the external primers BClibrary amp v2 and KpnI reverse v2.

TABLE 6 DNA oligonucleotides used for 309FGwt LO library generationOligo Sequence SEQ ID NO BC9 PCR5′-ACCGCGCTGATTACCTGGTCT1213111GGCTGTGAACTGACCTATGG 190 CATTAAAGATGBC 9-loop  5′-ACCGCGCTGATTACCTGGNNKNNKSMGNNKGSTNNKNNKNNKGGCTGT 191 NNKGAACTGACCTATGGCATTAAA-3′ 309 BC-loop5′-ACCGCGCTGATTACCTGG76K45K45K77K44K65K78T45K44KTGT 192 NNKdopeGAACTGACCTATGGCATTAAA-3′ DE PCR5′-GATGTGCCGGGCGATCGCACCACCATAGATCTG111111TATAGCATT 193GGTAACCTGAAACCGG-3′ Upstr BCloop CCAGGTAATCAGCGCGGTGGTAT 194 RevBC shuffle CAGATCTATGGTGGTGCGATCGC 195 rev DE shuffleTGTGAACTGACCTATGGCATTAAAGATGT 196 FWD 1 = Codons for all 19aa(-cys) 2= Codons for Ala/Pro 3 = Codons for Ala/Gly; 4 = 70%G10%A10%C10%T 5= 10%G, 70%A, 10%C, 10%T 6 = 10%G, 10%A, 70%C, 10%T 7 = 10%G, 10%A,10%C, 70%T 8 = 70%A15%C15%T K = 50%G/50%T

The NcoI-KpnI fragments were cloned into the phage display vector, andphage library generated as described in Example 1.

The four libraries were panned separately on Biotinylated HumanMegaCD40L as described for the first round in Example 2, using 4 μgCD40L in Round 1 and 1 μg in Round 2. After amplification of phageoutput after Round 2, single-stranded DNA was isolated using a QiagenSpin M13 kit (Qiagen, Valencia, Calif.), and the pools of BC-loopcontaining fragments from the BC loop libraries were amplified using BClib amp v2 and BC shuffle rev, whereas the pool of DE-loop containingfragments was amplified from the DE-loop library using primersDE-shuffle FWD and KpnI reverse v2. The PCR fragments were gel-purifiedand assembled through their overlapping sequence using the externalprimers BC lib amp v2 and KpnI reverse v2. The resulting PCR fragmentwas used to generate a library in the phage vector as previouslydescribed. This library was panned for a total of 5 rounds onbiotinylated Human MegaCD40L as described in Example 2, except thelibraries were initially contacted with a target at a concentration of50 nM, 20 nM, 20 nM, and 10 nM (in a total volume of 50 μl) for Rounds 1through 4 for 2 hours prior to incubation with blocked M280 streptavidinmagnetic beads for 10 minutes followed by washing.

Outputs were pool cloned into the NcoI-KpnI sites of plasmidpSec-oppA(L25M)-Tn3 pSec, and sixteen 96 well plates were screened forCD40L binding using soluble protein in the screening assay describedabove. The 270 highest scoring clones were cherry-picked, re-assayed andsequenced. Ten clones were chosen for further characterization based onbinding assay and sequence analysis. This included assessment of potencyin the PBMC assay, K_(d) determination for binding to CD40L in abiosensor assay, thermodynamic stability determined by differentialscanning calorimetry, and tendency to aggregate or form higher orderoligomers in solution by size exclusion chromatography analysis. Resultsare summarized in TABLE 7. Sequences of the 309 and 309FGwt clonesaligned with the ten optimized clones (designated 340, 341, 342, 343,344, 345, 346, 347, 348 and 349) are shown in FIGS. 11A and 11B.

Affinity matured variants showed 1-3 logs higher potency than the 309clone, retained high stability as measured by DSC, and most weremonodispersed as measured by SEC.

TABLE 7 Tm, DSC Variant PBMC IC50 (nM) Kd (nM) SEC Profile (° C.) 309226 191 OK 72 309FGwt 760 nd OK 71 340 0.7 2.2 OK 77 341 0.7 nd OK(?) 71342 0.7 1.4 OK 73 343 0.6 2.0 OK 69 (?) 344 1.3 nd OK (65 + 78.5) 34537.3 39 OK 72 346 9.0 14.9 OK 71 347 11.0 10.7 OK 70 348 1.0 1.8 ? nd349 38.2 21 OK(?) nd

PBMC assays were performed by stimulating PBMCs with humanCD40L-expressing Jurkat cells (D1.1, ATCC), and the percentage ofCD19+/CD86+ cells was measured by FACS after 24 hours. This assay wasused to test and rank the panel of leading Tn3 scaffolds to emerge fromprioritization based on biochemical criteria. Results of the PBMC assaysare shown in FIG. 10B, and summarized in TABLE 7.

Affinity measurements were performed on the ProteOn XPR36 proteininteraction array system (Bio-Rad, Hercules, Calif.) with GLC sensorchip at 25° C. ProteOn phosphate buffered saline with 0.005% Tween 20,pH 7.4 (PBS/Tween) was used as running buffer. Human MegaCD40L wasimmobilized on the chip at a surface density of approximately 2300 RU.Two-fold dilutions of the Tn3 variants (340, 342, 343, 345, 346, 347,348, and 349) were prepared in PBS/Tween/0.5 mg/ml BSA, pH 7.4 (from 150to 4.7 nM). Samples of each concentration were injected into the sixanalyte channels at a flow rate of 30 μl/min. for 300 seconds. The K_(d)was determined by using the equilibrium analysis setting within theProteOn software. Results are shown in TABLE 7.

The ten TCD40L-specific Tn3 variants were analyzed for stability by DSC.Briefly, DSC measurements were conducted on a VP-Capillary DSC(MicroCal). Proteins were exchanged into PBS (pH 7.2) through extensivedialysis, and adjusted to a concentration of 0.25-0.5 mg/ml for DSCanalysis. Samples were scanned from 20-95° C. at a scan rate of 90°C./hour, with no repeat scan. The results are shown in TABLE 7.

Up to a 300-fold improvement in IC₅₀ over 309, and over 1000-foldimprovement over the 309FGwt backbone used for lead optimization librarygeneration was obtained for the best clones. Seven clones had K_(d)'s inthe single digit nM range.

3.3 CD40L-Specific Tn3 Scaffold 311 Lead Optimization

Prior to conducting the loop usage experiment mentioned previously (seeFIG. 10A)

an initial attempt at a lead optimization library focused on introducingdiversity in the FG-loop of 311, generating and screening the resultingphage display library. Screening was conducted after 4 rounds of panningon Biotinylated human MegaCD40L as previously described, and it wasfound that a majority of the positive hits contained a fortuitousmutation of residue K4 to E in the N-terminal constant region of Tn3,upstream of the BC-loop. No obvious consensus among the FG-loopsequences of positive hits was detected. Introducing the single K4Emutation into 311 resulted in an approximately 100-fold increase inpotency (from approximately 4 μM to 36 nM) in the PBMC assay (see FIG.10C).

As the loop swap experiment indicated that the BC and DE loop sequenceswere required for binding to CD40L, these loops were then targeted inthe 311K4E backbone for further affinity maturation. Two separatelibraries were generated. One library targeted the BC-loop with astrategy where each residue had a 50% chance of being wild type 311sequence, and approximately 50% chance of being one of the other 11 NHTencoded residues. The other library completely randomized the 6-residueDE-loop.

For the BC-loop library, the oligonucleotides BC11-311Gly andBC11-311NHT (TABLE 8) were used in PCR reactions with the reverse primerKpnI amp rev v2 (TABLE 5) on a 311 derived template in which the BC-loopcodons had been replaced with stop codons to generate fragmentsincluding BC, DE and FG loops. Finally, amplification of a 1:1 mixtureof these fragments with the primers BC library amp K4E and KpnI amp revv2 gave the full length Tn3 library fragment.

The DE-loop library was generated as the 309FGwt DE-loop librarydescribed above, except a 311 derived template was used in the PCRreactions and the BC library amp K4E primer was used in the final PCRamplification.

TABLE 8 Oligos employed for 311K4E lead optimization library generation.Oligo Sequence SEQ ID BC11- 5′-ACCGCGCTGATTACCTGG26T25TV1T46T 197 311Gly46T45T45T25T37T35TGGCTGTGAACTGACC TATGGCATTAAA-3′ BC11-5′-ACCGCGCTGATTACCTGG26T25TV1T46T 198 311NHT46T45T45T25T37T35TNHTTGTGAACTGACC TATGGCATTAAA-3′ BC library5′-GGCCCAGCCGGCCATGGCCGCCATTGAAGT 199 amp K4EGGAAGATGTGACCGATACCACCGCGCTGATTAC CTGG-3′ BC11-5′-ACCGCGCTGATTACCTGG26T25TV1T46T 197 311Gly46T45T45T25T37T35TGGCTGTGAACTGACC TATGGCATTAAA-3′ BC11-5′-ACCGCGCTGATTACCTGG26T25TV1T46T 198 311NHT46T45T25T37T35TNHTTGTGAACTGACCTAT GGCATTAAA-3′ BC library5′-GGCCCAGCCGGCCATGGCCGCCATTGAAGT 199 amp K4EGGAAGATGTGACCGATACCACCGCGCTGATTAC CTGG-3′ 1 = 70%G, 10%A, 10%C, 10%T 2= 10%G, 70%A, 10%C, 10%T 3 = 10%G, 10%A, 70%C, 10%T 4 = 10%G, 10%A,10%C, 70%T 5 = 70%A, 15%C, 15%T 6 = 15%A, 70%C, 15%T 7 = 15%A, 15%C,70%T V = 33%A, 33%C, 33%G H = 33%A, 33%C, 33%T

The full length library fragments were digested with NcoI and KpnI,cloned into the phage display vector, and phage libraries generated asdescribed in Example 1.

The two libraries were panned separately on Biotinylated human MegaCD40Las described in Example 1, using 10 g of protein in Round 1, 5 g inRound 2, and 5 g in Round 3. After amplification of phage output afterRound 3, single stranded DNA was isolated using a Qiagen kit (Qiagen,Valencia, Calif.), and the two libraries were shuffled as described forthe 309FGwt libraries. The shuffled library was panned for a total of 5rounds on biotinylated human MegaCD40L as described above for the309FGwt shuffled library using 100 nM, 20 nM, 4 nM, 1 nM and 1 nM oftarget for Rounds 1 through 5.

Outputs were pool cloned into the soluble secretion vector as previouslydescribed, and five 96-well plates were screened for CD40L binding usingthe soluble protein screening assay described previously. Positive hitswere identified relative to the signal obtained with the 311K4E backbonevariant. The 18 highest scoring unique clones, designated 311K4E_1,311K4E_2, 311K4E_3, 311K4E_4311K4E_5, 311K4E_7, 311K4E_8, 311K4E_9,311K4E_10, 311K4E_11, 311K4E_12, 311K4E_13, 311K4E_14, 311K4E_15,311K4E_16, 311K4E_19, 311K4E_20 and 311K4E_21 (sequences shown in FIG.12A and FIG. 12B) were assayed as crude unpurified proteins for off-rateranking. Off-rate estimates of unpurified Tn3 scaffolds were performedon the ProteOn XPR36 protein interaction array system (Bio-Rad,Hercules, Calif.) in a biosensor assay with CD40L immobilized on a chip.Mega human CD40L was immobilized on a GLC chip (BioRad) and all variantsdiluted to an estimated concentration of 80 nM, injected at a flow rateof 30 μl/min for 300 seconds with the dissociation time set to 1200seconds. PBS, 0.005% Tween20, 3 mM EDTA, pH7.4 was used as runningbuffer. Off-rates were ranked by visual inspection of the sensorgrams. Asubset of four variants which displayed the slowest off-rates, 311K4E_3,311K4E_11, 311K4E_12 and 311K4E_15, was purified, and K_(d) values weredetermined to be between 1.1 and 6.4 nM (TABLE 9).

TABLE 9 K_(d) of 311K4E and 4 affinity purified variants binding tohuman CD40L. 311 Variant K_(d) (nM) 311K4E 18 311K4E_12 1.1 311K4E_116.3 311K4E_15 1.6 311K4E_3 6.4

As indicated in FIG. 10D, the decreased K_(d) (from 18 nM to 1 nM) of311K4E_12 corresponded to a 12-fold increase in potency in the PBMCassay relative to the 311K4E backbone.

In conclusion, the lead optimization campaigns of initial hits 309 and311 from the naïve libraries lead to single-digit nM binders of CD40L.

A similar optimization campaign was performed on the murine-specific M13molecule (data not shown). The resulting optimized murine CD40L-specificmolecule (designated M31) showed an approximately 20-fold increase inpotency in the PBMC assay over the parent molecule (FIG. 1A).

Example 4 Expression and Purification of Untagged CD40L-Specific Tn3-HSAFusions

Tn3 constructs fused to HSA as outlined in FIGS. 2A and 9A wereexpressed in mammalian 293F cell line by transient transfection. Tn3-HSAfusion expression constructs were generated based on an in-housegenerated mammalian expression vector. To increase product homogeneity,a mutant form of HSA (designated HSA C34S) was employed in which theunpaired, partially exposed cysteine 34 had been mutated to serine (Zhaoet al., 2009, Eur. J. Pharm. Biopharm. 72: 405-11).

The fusion protein could be purified in a one-step purification by ionexchange chromatography (IEX). An example of elution of 309-309-HSA froma Q-HP column (GE HealthCare) by a salt gradient is shown in FIG. 9B. Inaddition to the main peak, minor later eluting peaks were seen(constituting less than 10% of the total peak area). Mass spectrometryanalysis indicated these minor side peaks were enriched forO-glycosylated 309-309-HSA species. Fractions containing the main peakwere pooled and used for subsequent activity assays.

For larger scale purifications, the IEX step mentioned above waspreceded by capturing the Tn3-HSA fusions from the culture supernatantby affinity chromatography using HSA affinity matrixes, e.g., HiTrapBlue HP (GE HealthCare). After washing, HSA fusion protein could beeluted with an Octenoic Acid containing buffer. Eluate was loaded ontothe Q-HP column after 3-fold dilution in phosphate buffer.

Analysis of the minor peak(s) revealed the presence of O-linkedcarbohydrate moieties. The O-glycan were proposed to be a heterogeneousmix of carbohydrates derived from a previously reported O-xylosylatedcore structure (Wakabayashi et al., 1999, J. Biol. Chem. 274:5436-5442)shown below:

The predominate site of the attachment was determined to be in the GGGGS(SEQ ID NO: 147) linker between the Tn3 domains. The glycan was alsofound to be present to a lesser extent in the GGGGS (SEQ ID NO: 147)linker found between the Tn3 and HSA. The levels of O-glycan weretherefore higher in the bivalent constructs as compared to themonovalent constructs and were higher in material produced in HEK cellsas compared to CHO cells. The levels also varied between the differentTn3 constructs. Thus, the level of O-glycan may be reduced throughcareful host cell section, for example use of CHO cells or other cellsfound to produce material with lower levels of O-glycan. In addition,material containing the O-glycan can be removed via purification methodsto yield a more homogenous product lacking the O-glycan. Alternatively,the linker may be modified to remove the primary site(s) of O-glycanattachment, for example by mutating the Ser residue to a Gly. Thelinkers in several constructs were reengineered to have one or moreGGGGG (SEQ ID NO:148) linker. No O-glycans of any type were detected inmaterial having the GGGGG (SEQ ID NO: 148) linker(s) and no differencein activity was seen (data not shown).

Example 5 Extension of Serum Half-Life of CD40L-Specific Tn3 Scaffolds

Fusion to serum albumin was explored as a strategy to extend the serumhalf-life of CD40L-specific Tn3 scaffolds. In order to determine thepharmacokinetic (PK) properties of murine CD40L specific Tn3-MSA fusionsa mouse PK assay was conducted. MSA fusions were chosen for studies ofsurrogate molecules over the corresponding HSA fusions since mouse FcRnbinds HSA considerably weaker than it binds MSA, resulting in decreasedrecycling from endosomes and consequently increased turnover (Andersenet al. J. Biol. Chem. 285, 4826-4836, 2010).

HEK 293 cells were used for expression of mouse CD40L-specific tandembivalent Tn3 scaffold fused to MSA. High levels of expression wereobserved (FIG. 3A). These constructs had a (G₄S) linker between the Tn3scaffold units and 3 (G₄S) repeats in the linker between the scaffoldand MSA. In addition, a N49Q mutation was introduced into each of theM13 and M31 scaffolds to remove a potential N-linked glycosylation site.This mutation did not affect the potency of these scaffolds (data notshown). Expression level was estimated to 200 mg/L 6 days posttransfection. Purification was carried out by IMAC through a C-terminalHis-tag. The yield of purified protein was estimated to be 125 mg/Lculture supernatant.

When MSA was fused to bivalent M13 scaffolds, an 8-fold decrease inpotency compared to the M13 dimeric scaffold without MSA was observed(FIG. 3B). A bivalent scaffold comprising affinity matured M31 fused toMSA was 140 times more potent than the corresponding bivalent M13scaffold fused to MSA, approximately 900 times more potent than themonovalent M31 MSA fusion, and had a potency comparable to the MR1 antimurine CD40L monoclonal antibody (FIG. 3C).

To determine the PK properties of CD40L-specific Tn3-MSA fusions a mousePK analysis was carried out. Protein constructs were administeredintravenously at 10 mg/kg in 5-7 week old female CD-1 mice. Each mousewas bled 150 μl at various time points and serum concentration ofTn3-HSA fusion determined by an ELISA assay. Briefly, Nunc MaxiSorpplates were coated with anti-FLAG M2 antibody (Agilent), blocked in 4%milk in PBS+0.1% Tween (PBST) and incubated with murine MegaCD40L(Axxora). The MegaCD40L was immobilized through its FLAG tag. Serumsamples and protein standards were diluted in 4% milk PBST and addedafter washing plate in PBST. After incubation the plate was washed inPBST and rabbit anti-TN3 polyclonal antibody (Covance) was used todetect the Tn3-HSA fusion constructs using Goat anti-rabbitHRP-conjugated antibody (Jackson ImmunoResearch) in a standard ELISAprotocol. Concentrations in serum samples were determined based onlinear regression of standard curves generated by assays of dilution ofthe same Tn3-MSA fusion construct. Concentrations were determined as anaverage for 3 different mice.

As seen in FIG. 4A, M31-MSA and M13-M13-MSA had half-lives of 38 and 31hours, respectively, whereas the M31-M31-HSA construct had a half-lifeof 12 hours. In comparison, the M13-M13 tandem construct by itself (notfused to MSA) displayed a half-life of 30 minutes (not shown).

In contrast to the observations in mouse scaffolds, when HSA was fusedto human CD40L specific scaffolds there was no significant decrease inpotency. FIG. 9C shows that there was no significant decrease in potencyby fusing a human CD40L-specific bivalent Tn3 scaffold comprising two309 monomer to HSA as measured in PMBC assays.

The PK properties of the human CD40L-specific 342-HSA monomer constructwere compared to those of a 342-HSA variant comprising two substitutions(L463N and K524L) to enhance serum half-life in Cynomolgus monkeyfollowing a single intravenous injection. Protein constructs wereadministered via slow bolus injection at 10 mg/kg to male Cynomolgusmonkeys weighing 2-5 kg. 1 mL of blood/animal/time point was collectedfrom a peripheral vessel at predose, 5 minutes and 30 minutes post dose;2, 12, 24, and 48 hours post dose; and on Days 4, 8, 11, 15, 22, 29, 36,43 and 57. Serum concentration of Tn3-HSA fusion determined by an ELISAassay. Briefly, Nunc MaxiSorp plates were coated with anti-FLAG M2antibody (Agilent), blocked in 4% milk in PBS+0.1% Tween (PBST) andincubated with human MegaCD40L (Axxora). The MegaCD40L was immobilizedthrough its FLAG tag. Serum samples and protein standards were dilutedin 4% milk PBST and added after washing plate in PBST. After incubationthe plate was washed in PBST and rabbit anti-TN3 polyclonal antibody(Covance) was used to detect the Tn3-HSA fusion constructs using Goatanti-rabbit HRP-conjugated antibody (Jackson ImmunoResearch) in astandard ELISA protocol. Concentrations in serum samples were determinedbased on linear regression of standard curves generated by assays ofdilution of the same Tn3-HSA fusion construct. Concentrations areplotted in FIG. 4B. The half-life of the 342-HSA construct was about 7days, while the 342-HSA L463N/K524L variant construct showed anincreased half-life of 13-17 days during the initial linear phase (FIG.4B). After 30 days, the serum concentrations of the 342-HSA L463N/K524Lvariant construct dropped off more rapidly as compared to the wild typeHSA construct. These observations may indicate some immunogenicity ofthis construct in monkeys.

Example 6 Characterization of CD40L-Specific Tn3 Scaffolds

6.1 Experimental Methods

6.1.1 PBMC Stimulation Assay:

Blood was obtained from healthy donors according to MedImmune safetyguidelines. PBMCs were isolated via CPT tubes (spin 1500 g for 20minutes) and 1×10⁶ PBMCs (per condition) were stimulated by recombinanthuman MegaCD40L (Axxora) or human CD40L expressing Jurkat cells (D1.1,ATCC). The percentage of CD19+/CD86+ cells was measured by FACS after 24hours stimulation. This assay was used to test and rank the panel ofleading Tn3 scaffolds to emerge from prioritization based on biochemicalcriteria. The assay can also be performed with a murine CD40L-expressingcell line (D10.G4) or murine MegaCD40L (Axxora ALX522120) in place ofthe human cell or recombinant protein stimulation as murine ligand crossreacts with human receptor.

6.1.2 MurineCD40R/NFκB Assay:

NFκB reporter NIH3T3 cells (Panomics NFκB reporter system and in-housemCD40R transfection) were stimulated with murine MegaCD40L (Axxora, cat.ALX522120) recombinant protein or CD40L over-expressing D10.G4 cells(ATCC) for 24 hours with or without Tn3 scaffolds. Bright-Glow (PromegaE2610) was added according to manufacturer's directions. The readout wasluminescence (700) via the NFκB reporter activation performed on anEnVision system (Perkin Elmer).

6.1.3 Human CD40R/NFκB Assay:

Reporter HEK293 cells (Panomics and in-house) were stimulated withMegaCD40L (Axxora ALX522110) recombinant protein or CD40L overexpressingD1.1 Jurkat subclone cells (ATCC) for 24 hours with or without Tn3scaffolds. Bright-Glow (Promega E2610) was added according tomanufacturers directions. The readout was luminescence (700) via theNFκB reporter activation performed on an EnVision system (Perkin Elmer).

6.1.4 Dual Cell Assay:

Primary T/B cells were isolated from various donors. Anti-CD3stimulated, mitomycin C treated human CD4+ T cells (1×10⁵) were culturedwith purified human B cells (5×10⁴). Readouts were as follows: Day 2:Activation markers (FACS), Day 5: B cell proliferation (ATP metabolite,Cell-Titer Glo, Invitrogen), Day 7: plasma cell differentiation (FACS)Day 7: Ig production (ELISAs, R&D Systems).

6.1.5 Platelet Aggregation Assays:

Adenosine diphosphate (ADP) was from Chrono-Log (Havertown, Pa., USA).All other products were at least reagent grade. Blood samples werecollected from healthy volunteers in 12.9 mM sodium citrate andcentrifuged at 150×g for 15 minutes to obtain PRP (platelet richplasma). After separation of PRP, tubes were centrifuged again at1,200×g for 15 minutes to obtain PPP (platelet poor plasma). Plateletswere washed using the method described by Mustard et al. (Br. J.Haematol. 22:193-204, 1972), and re-suspended in Tyrode's solutioncontaining CaCl₂ 2 mM, MgCl₂ 1 mM, 0.1% dextrose, 0.35% bovine serumalbumin, 0.05 U/mL apyrase, pH 7.35. Platelet aggregation was studiedusing a light transmission aggregometer (Chrono-Log 700-4DR, Chrono-LogCorporation, Havertown, Pa., USA) and recorded for 10 min afterstimulation of platelets with the indicated platelet agonists asdescribed. Tn3 scaffolds were pre-incubated with the soluble CD40L(sCD40L) to form immunocomplexes prior to addition.

6.1.6 Immunization Assays:

Sheep Red Blood Cells (SRBC) were purchased from Colorado Serum (Denver,Colo.) and diluted 10-fold in HBSS medium immediately before use. Micewere immunized with 0.2 ml of diluted SRBC on day 0. Primary Germinalcenter (GC) response in challenged mice was assayed 14 days afterimmunization via FACS (GC B cells, non-GC B-cells, and all T cellsubsets). Tn3 scaffolds and controls were administered on days 9-13 in24 hour increments as indicated.

6.1.7 KLH-Specific T Cell Dependent Antibody Response (TDAR) Assays:

Cynomolgus Monkeys (Macaca fascicularis) of Chinese origin and weighing3.1-4.6 kg, (Covance Research Products, Alice Tex.) were dosedintravenously (saphenous or cephalic vein) once weekly with theindicated dose (0.5, 5, 40 mg/kg) of inhibitor (342-monomer-Tn3-HSA and342-342 bivalent-Tn3-HSA or control/vehicle. KLH (Lot No. MD158678A,Supplier Pierce Biotechnologies, Rockford, Ill.) was reconstituted withthe appropriate amount of sterile water for injection (Supplier MidwestVeterinary Supply, Norristown, Pa.) under sterile conditions. Vials wereswirled to mix and then pooled together into a sterile vial. 1 mL of KLHsolution (10 mg/mL) was administered subcutaneously on each animal'sback, to the left of the midline on two occasions (Day 1 and Day 29),within 1 hour of the end of the test or control article administration.Blood samples for further analysis were obtained from all animals at thefollowing time points: pretest, Days 4, 6, 8, 11, 15, 22, 25, 32, 34,36, 39, 43, 46, 50 and 57. Samples collected on Days 8, 15 and 22 weretaken prior to dosing. Evaluation of KLH-specific IgM and IgG antibodytiters were done at days 8, 11 and 15. The titers of KLH-specific IgMand IgG antibodies at day 15 are shown in FIGS. 5G and 5H, respectively.The cutpoint titration method utilized an ELISA format to detectanti-KLH antibodies in monkey serum. The samples were incubated withKLH, which was immobilized on an ELISA plate. After incubation, theplates were washed, and the bound antibodies were detected with Goatanti-Monkey IgG-HRP or IgM-HRP and then visualized withtetramethylbenzidine (TMB).

For all experiments utilizing animals currently acceptable practices ofgood animal husbandry were followed e.g., Guide for the Care and Use ofLaboratory Animals; National Academy Press, 2011. Huntingdon LifeSciences, East Millstone, N.J. is fully accredited by the Associationfor Assessment and Accreditation of Laboratory Animal Care International(AAALAC). Animals were monitored by the technical staff for anyconditions requiring possible veterinary care and treated as necessary.

6.2 CD40L specific Tn3 Scaffolds Functionally Neutralize CD40L: CD40Interactions.

CD40L-expressing T cells engage CD40-expressing B cells resulting in theactivation of NFκB signaling pathway (Zangani, 2009). Thus, anNFκB-luciferase reporter cell line was used to determine if theanti-CD40L-Tn3 molecules could inhibit signaling downstream of CD40engagement. HEK293 cells expressing human CD40L and the reporter werestimulated with either human or murine MegaCD40L at EC₉₀ (EffectiveConcentration that results in 90% inhibition; i.e., 1.5 μg/ml for humanMegaCD40L, and 3 μg/ml for murine MegaCD40L).

The human-specific 342 molecule inhibited human CD40L-induced NFκBactivity with an IC₅₀ of 1.5 nM (FIG. 13 ). The murine-specific M31molecule, neutralized murine CD40L-induced NFκB activity with anIC₅₀=1.6 nM (FIG. 1B). The positive control anti-CD40L monoclonalantibodies 5c8 (anti human CD40L) and MR1 (anti murine CD40L) bothperformed about 10-fold better than the monomeric Tn3 scaffolds withIC₅₀'s of 0.200 nM+/−SD (lowest threshold of the assay). This could bein part due to the bivalent nature of the monoclonal antibodiescontributing to the avidity of the interaction with their respectiveCD40L's.

6.3 Dimeric CD40L Specific Tn3 Scaffolds Exhibit Improved Binding.

Experimental data indicates that the binding of a CD40L-specific Tn3bivalent scaffold was improved over that of a CD40L-specific Tn3 monomerscaffold. The binding of the CD40L-specific Tn3 bivalent scaffold toCD40L improved the action on the target, in some cases by approximately3 logs over that of a CD40L specific Tn3 monomer scaffold in vitro, asshown in FIG. 2C and FIG. 2D (murine), and FIG. 8A and FIG. 8B (human).

FIG. 2C shows the competitive inhibition of murine CD40L binding tomurine CD40 receptor immobilized on a biosensor chip by murineCD40L-specific monovalent (M13) or bivalent tandem scaffolds. M13monomers were linked with varying length peptide linkers comprising one(1GS), three (3GS), five (5GS) or seven (7GS) “GGGGS” (SEQ ID NO: 147)repeats. The IC₅₀ of the M13-1GS-M13 scaffold was 29 nM, whereas theIC₅₀ of the monomer M13 scaffold was 71 nM. The IC₅₀ of divalent M13scaffolds with longer linkers were dramatically lower (5-6 nM).

FIG. 2D shows the inhibitory effect of murine CD40L-specific monovalent(M13) or bivalent tandem scaffolds on murine CD40L-induced CD86expression on B cells. The bivalent scaffolds were approximately 3 logsmore potent than the monovalent scaffolds.

FIGS. 8A and 8B show that human CD40L-specific Tn3 scaffolds 309 and 311displayed enhanced potency in a bivalent tandem format. The bivalent 311scaffolds (FIG. 8A) and the bivalent 309 scaffolds (FIG. 8B) showedapproximately a 7-fold and a 500-1000-fold improvement, respectively, ininhibition of human CD40L-induced expression on CD19 positive humanPBMCs stimulated with Jurkat D1.1 cells. The bivalent 309 scaffolds werecomparable in potency to Biogen's 5c8 anti human CD40L monoclonalantibody.

Solubility, stability and ease of purification was not disrupted withthe addition of varying length peptide linkers comprising one (1GS),three (3GS), five (5GS) or seven (7GS) “GGGGS” (SEQ ID NO: 147) repeats(see FIG. 2B).

6.4 CD40L Specific Tn3 Scaffolds Binding and Function.

In addition to the biochemical binding described above, it was importantto verify that these novel Tn3 scaffolds were able to bind endogenousCD40L expressed on primary T cells following activation. T cells wereisolated from multiple donors and activated as described. After 24hours, CD40L expression was upregulated as determined by staining with5c8 (human-specific) monoclonal antibody and MR1 (murine-specific)monoclonal antibody (data not shown). The CD40L specific Tn3 scaffoldmolecules were able to detect comparable levels of CD40L expression asthe monoclonal antibodies confirming that these molecules can bindnative protein.

One of the functional consequences of the CD40L:CD40 interaction is theup regulation of co-stimulatory molecules on B cells (Yellin et al., J.Exp. Med. 6:1857-1864, 1995). The CD40L-directed Tn3 molecules weretested for their ability to prevent this. Cell lines endogenouslyexpressing human or murine CD40L (D1.1 Jurkat subclone or D10.G4respectively) were used to stimulate peripheral blood mononuclear cells(PBMC). Once stimulated, the activation of B cells was assessed bymeasuring the percentage of CD86 up regulation by CD19+ B cells via flowcytometry. In this assay, the positive control monoclonal antibodieswere able to inhibit the CD19+ percentage of cells with CD86 expressionwith IC₅₀s of 0.170 nM (5c8) and 0.230 nM (MR1). The human-specificoptimized Tn3, 342 was able to antagonize CD86 up regulation with IC₅₀values=0.700 nM (n=5 donors) (see FIG. 10B and TABLE 7).

The murine-specific optimized Tn3 M31 had and IC₅₀ of 1.5 nM. Thesesimilar results were observed when Mega-CD40L recombinant protein wasused to stimulate PBMCs. The experimental data demonstrated that bothmolecules, whether murine or human specific, cannot only inhibit themain signaling pathway within a cell (NFκB), but also one of its mostimportant functional roles: T-B cell interactions. This inhibitoryaction can counteract CD40L's contribution in many auto immune diseasesand conditions.

6.5 Anti-CD40L Tn3's Inhibit B Cell Proliferation and Plasma CellDifferentiation Following T/B Co-Culture.

Interactions of CD40L on T cells with CD40-expressing B cells are afundamental aspect of T cell help which facilitates the development ofadaptive immune responses (Banchereau, 1994; Oxenius, 1996, van Kooten &Banchereau, 1997). To model this, the anti-hCD40L Tn3-HSA fusions of340, 342 and 342-342 dimer were evaluated in primary cell co-cultures ofT cells and B cells where anti-CD3 stimulated, mitomycin C treated humanCD4+ T cells were cultured with purified human B cells. The ability ofthe B cells to proliferate at day four to differentiate into plasmacells (PC) by day seven and switch their antibody class of productionwere measured (PC and antibody data not shown) (FIG. 15 ) (Ettinger,2007). The CD40L-specific Tn3 scaffold 342-342-HSA was able to reduce Tcell induced proliferation by at least 50% as compared to the cellproliferation of B-cells in the absence of scaffolds, or in the presenceof a non-specific control scaffold. Proliferation is a pre-cursor,signal one, to plasma cell differentiation, upon CD40L:CD40 ligation.Inhibition of plasma cell differentiation and antibody class switching(data not shown) were also observed.

6.6 In Vivo Disruption of the CD40:CD40L Axis.

The central role of CD40L:CD40R interactions in T-dependent immuneresponses have been well characterized (Noelle, 1992; Renshaw, 1994,Wykes, 2003). The murine CD40L-specific Tn3 scaffold M31 (M31-MSA andM31-M31-MSA) was used to evaluate the effects of these novel moleculesin a T-dependent immunization model by immunizing mice (intravenously)at day zero with Sheep Red Blood Cells (SRBC).

On days 9-13, mice were injected intraperitoneally daily with theindicated dose of inhibitor and at day 14 splenic and lymph node GC Bcells were quantitated. Daily dosing was required given the shortT_(1/2) of this molecule in vivo, 31 hours (FIG. 4 ). It is wellestablished that CD40L controls humoral responses such as the generationof germinal centers in anatomical sites such as the spleen and lymphnodes from previous findings (Jacob, 1991). Here, the disruption of theCD40L:CD40 axis contribution to that formation was observed in a dosedependent manner with M31-MSA versus naïve or our non-specific control,D1-MSA, as shown by the percentage of GC B cells.

Even at 10 mg/kg M31-MSA was able to abolish the percent of GC B cells(FIG. 5B) as well as the MR1 monoclonal antibody (FIG. 5A). Othersub-populations of cells appeared normal including specific T-cellpopulations assuring that the results observed were not due to T celldepletion (FIG. 5C, FIG. 5D, FIG. 5E). In addition, the results from theanti-SRBC Ig ELISA data mirrored those of the germinal center B celldata (FIG. 5F). Taken together, these data indicated that themurine-specific Tn3 scaffold M31-MSA can abrogate reactions driven viaCD40 signaling.

Similarly, the human CD40L-specific Tn3 scaffold 342 (342-HSA and342-342-HSA) was used to evaluate the effects of these novel moleculesin KLH-specific T cell dependent antibody response (TDAR) model inCynomolgus Monkeys. Here, the disruption of the CD40L:CD40 axis resultsin suppression of antibody generation to the KLH antigen in a dosedependent manner. As shown in FIGS. 5G and 5H the 342-bivalent constructsuppressed the levels of IgM and IgG antibodies at 0.5 mg/kg (mpk) andnearly complete suppression was seen at 5 mg/kg. The 342-monomerconstruct also suppressed the levels of IgM and IgG but at higherconcentrations with nearly complete suppression seen at 40 mg/kg. Thesedata indicated that the human-specific Tn3 scaffold constructs 342-HSAand 342-342-HSA can both abrogate reactions driven via CD40 signaling.

6.7 Human CD40L-Specific Tn3 Scaffolds do not Induce PlateletAggregation.

Human clinical trials with anti-CD40L monoclonal antibodies were haltedwhen thromboembolisms occurred in several patients (Davidson et al. ArthRheu, 43:S271). Subsequent pre-clinical analyses suggested this to be anon-target class effect of anti-CD40L monoclonal antibodies. Thus it wasimportant to test the human CD40L-specific Tn3 scaffolds in plateletaggregation assays.

When a ratio of three molecules of physiological CD40L to one moleculeof the anti-CD40L monoclonal antibody was used, pro-aggregator effectswere observed in citrated Platelet Rich Plasma (PRP), washed platelets,and whole blood (FIG. 16A). These effects were mediated by monoclonalantibody Fc domain dependent interactions subsequent to CD40L binding(data not shown). In the absence of the Fc domain fusion, no aggregationwas observed. No aggregation was observed in multiple donors with any ofthe human CD40L specific Tn3 scaffolds either as dimers or as HSA fusionproteins (FIG. 16B and FIG. 16C).

Deleterious side-effects as observed in the clinical trials wereobserved by creating a soluble CD40L/anti-CD40L monoclonal antibodyimmunocomplex in the presence of platelets (FIG. 16A and 5C8 trace onFIG. 16C). Another example of this can be seen in the histology of thetransgenic human FcγRIIa murine study (Francis et al., 2010). Uponadministration of soluble CD40L/monoclonal antibody immune complexes, anabundance of thrombi was seen within the lung tissue within minutesafter administration. However, when duplicated with anti-CD40L Tn3scaffolds, normal histology was present in the lung in accordance withthe control samples (data not shown).

Example 7 Fibronectin Type III Domains Engineered to Bind CD40L:Cloning, Expression, Purification, Crystallization and Preliminary X-RayDiffraction Analysis of Two Complexes

Recombinant human soluble CD40L was co-crystallized with twoCD40L-specific Tn3 monomer scaffolds, 309 and 311K4E-12, both isolatedas CD40L binders from phage display libraries. The crystals diffractedto 3.1 and 2.9 Å respectively. In addition, recombinant human solubleCD40L was co-crystalized with the optimized Tn3 monomer 342 alone andwith both the 342 monomer and the 311K4E_12 monomer. The crystals forthese structures diffracted to 2.8 and 1.9 Å respectively. Thecorresponding crystal structures help to understand the interactionbetween Tn3 scaffolds and CD40L and can be used to design higheraffinity CD40L binders and tandem constructs binding multiple epitopes.

7.1 Expression and Purification of Tn3 Molecules and Human Soluble CD40L

To produce tagless Tn3 molecules for crystallization, the proteins wereexpressed in E. coli using an in-house IPTG-inducible vector designed tosecrete recombinantly expressed proteins into the periplasmic space.This vector has a Ptac promoter, OppA signal peptide mutant L25/M(MTNITKRSLVAAGVLAALMAGNVAMA) (SEQ ID NO: 210), a C-terminal 8×His-tag inaddition to a thrombin cleavage site. The Tn3 sequences were subclonedbetween signal peptide and thrombin cleavage site.

Expressed secreted His-tagged proteins were purified using Ni-NTA resinaccording to the manufacturer's instructions (Qiagen, Valencia, Calif.,USA) and then cleaved by thrombin followed by Ni-NTA affinitypurification again to remove the uncut intact protein and the cutHis-tagged fragment. This purification step was followed by ion-exchangestep using HiTrap Q columns (GE Healthcare, Piscataway, N.J., USA)performed on Äkta Purifier (GE Healthcare, Piscataway, N.J., USA). Thepurified tagless Tn3 proteins show greater than 95% purity andhomogeneity based on SDS-PAGE and SEC results.

Human soluble CD40L (113-261, UNIPROT: P29965) gene was synthesized byGeneArt with an N-terminal 6×His-tag and was cloned into an in housemammalian expression vector under the control of the cytomegalovirusmajor immediate early (hCMVie) promoter (Boshart et al., Cell 41:521-530, 1985). The CD40L gene was cloned in frame with a CD33 signalpeptide. The EBNA and Ori P genes in the vector were used to increaseprotein expression. The CD40L gene also incorporated a SV40 poly-Asequence to allow proper processing of its mRNA 3′-end. The constructwas transiently transfected into 293F suspension cells (human embryonickidney cells [HEK] grown in 293 Freestyle Medium and using 293 Fectin,Invitrogen, Carlsbad, Calif., USA). Cells were grown using standardprotocol and media was harvested after 4 and 8 days. The soluble CD40Lprotein was then purified using Ni-NTA resin followed by an ion-exchangestep using Hi-Trap SP FF column (GE Healthcare, Piscataway, N.J., USA)and dialysis against 50 mM Tris pH 7.5, 50 mM NaCl.

To prepare complexes the Tn3 molecule, either 309 or 311K4E_12 or 342,was mixed with CD40L in a 1.1:1 ratio, concentrated using Vivaspinconcentrators (30,000 Da cut-off; GE Healthcare, Piscataway, N.J., USA)to approximately 10 mg/ml and subjected to size-exclusion chromatography(SEC) using Superdex 200 10/300 GL column (GE Healthcare, Piscataway,N.J., USA) pre-equilibrated with 50 mM Tris-HCl, pH 7.5, 100 mM NaCl,0.02% NaN3 (FIG. 19 , panel A). After the separation step the complexwas concentrated to 18 mg/ml and subjected to crystallization. The342-311K4E_12-CD40L complex was prepared essentially as described aboveusing a 1.1:1.1:1 ratio of the three components.

7.2 Crystallization Screening and Optimization

Sitting drop crystallization experiments were set up in 96-wellIntelli-plates (Art Robbins Instruments, Sunnyvale, Calif., USA) using aPhoenix crystallization robot (Art Robbins Instruments, Sunnyvale,Calif., USA) by mixing 300 nL volumes of well solution and proteincomplex solution in the drop compartment and letting it equilibrateagainst 50 μL of well solution. Commercial crystallization screens fromHampton Research (Aliso Viejo, Calif., USA), Emerald BioSystems(Bainbridge Island, Wash., USA) and Molecular Dimensions (Apopka, Fla.,USA) were used.

Crystallization of the 309-CD40L, 342-CD40L and 342-311K4E_12-CD40Lcomplexes each required an optimization step which included additionalscreening using Additive Screen HT (Hampton Research, Aliso Viejo,Calif., USA). In the optimization step the well solution of the 96-wellplate was filled with 80% of successful solution from the initialscreening and 20% of respective additive. The drop was made of 300 nL ofprotein solution and 300 nL of the new well solution after thoroughmixing of the latter. The diffraction quality crystals were harvesteddirectly from 96-well plate. For cryo-preservation the crystal wastransferred into three consecutive solutions of mother liquor withincreasing glycerol concentrations.

The diffraction quality 311-CD40L crystals grew at the initial screenout of solution that did not require addition of cryo agent.

7.3 X-Ray Diffraction and Data Collection

X-ray diffraction patterns for the 309-CD40L complex were collected fromsingle crystal at the Beamline 5.0.3 of the Advanced Light Source inLawrence Berkeley National Laboratory (University of California,Berkeley) equipped with ADSC Q315R CCD X-Ray detector (Area DetectorSystems Corporation, Poway, Calif., USA). 360 consecutive images withoscillation range of 0.5° were collected at crystal-to-detector distanceof 300 mm and an exposure time of 0.8 seconds.

X-ray diffraction patterns for the 311K4E_12-CD40L, 342-CD40L and342-311K4E_12-CD40L complexes were collected from single crystals at theBeamline 31-ID-D of the Advanced Photon Source in Argonne NationalLaboratory (University of Chicago, Chicago, Ill.) equipped with aRayonix 225 HE detector (Rayonix LLC, Evanston, Ill., USA). 180consecutive images with oscillation range of 10 were collected atcrystal-to-detector distance of 300 mm and an exposure time of 0.8seconds.

Reduction and scaling for all data sets were performed using HKL2000suite (Otwinowski & Minor, Methods in Enzymology, 276:307-326. 1997).

7.4 Results and Discussion

The most reproducible crystallization condition of the 309-CD40L complexappeared to be B5 (0.2 M NaNO₃, 20% PEG3350) in Peg/Ion Screen (HamptonResearch). Further optimization with Additive Screen yielded diffractionquality crystal from the A1 condition (0.1M BaCl2·2H2O). The crystalshown in FIG. 19 , panel B was harvested from a 96-well plate, andcooled in liquid Nitrogen after transfer to the mother liquor solutionsupplemented with 20% Glycerol.

Space group symmetry: The crystal belonged to orthorhombic space groupP212121 with cell parameters a=85.69 Å, b=90.64 Å, c=95.56 Å anddiffracted to 3.1 Å. The asymmetric unit is expected to contain a trimerof CD40L and three 309 molecules with VM value about 2.3 Å3/Da.

For the 311K4E_12-CD40L crystallization the Cryo I & II screen (EmeraldBioStructures) yielded number of conditions which required neitheroptimization nor cryo preservation. A single crystal (FIG. 19 , panel C)from condition F7 (40% PEG 600, 0.1M CH3COONa, 0.2M MgCl2) was used fordata collection.

Space group symmetry: The crystal belonged to cubic space group P213with cell parameter 97.62 Å and diffracted to 2.6 Å. The asymmetric unitcontains one CD40L and one 311K4E_12 molecule with VM value about 2.9Å3/Da.

342-CD40L space group symmetry: The crystal belonged to space group P321with cell parameters a=93.53 Å, b=93.53 Å, c=66.69 Å, resolution 2.8 Å.The asymmetric unit contains one CD40L monomer and one 342 monomer.

342-311K4E_12-CD40L space group symmetry: The crystal belonged to spacegroup P21 with cell parameters a=80.32 Å, b=143.48 Å, c=111.27 Å,β=98.22°, resolution 1.9 Å. The asymmetric unit contains two CD40trimers, six 342 monomers, and six 311K4E-12 monomers.

Data statistics for all the structures are shown in TABLE 10.

TABLE 10 X-Ray data collection statistics. 309-CD40L 311K4E-12Wavelength, {acute over (Å)} 0.9793 0.9793 Resolution, {acute over (Å)}50.0-3.05 (3.16-3.05) ^(a) 50.0-2.94 Space group P2₁2₁2₁ P2₁3 Cellparameters, {acute over (Å)} a = 85.69, b = 90.64, c = 95.56 a = 97.62Total reflections 94,024 128,140 Unique reflections 14,555 6720 Averageredundancy 6.5 (6.4) ^(a) 19.2 (19.7) Completeness, % 100.0 (100.0) ^(a)99.4 (100.0) R_(sym) 0.097 (0.443) ^(a) 0.114 (0.785) Mean I/σ (I) 17.2(4.6) ^(a) 20.1 (2.4) 342-CD40L 342-311K4E_12-CD40L Wavelength, {acuteover (Å)} 0.9793 0.9793 Resolution, {acute over (Å)} 50.0-2.8(2.83-2.82) ^(a) 144.5-1.9 (1.96-1.95) ^(a) Space group P321 P2₁ Cellparameters, {acute over (Å)} a = 93.53, b = 93.53, a = 80.32, b =143.48, c = 66.69 c = 111.27, β = 98.22° Total reflections 66,038 (549)^(a) 733,814 (1806) ^(a) Unique reflections 8,406 (88) ^(a) 179,232(1806) ^(a) Average redundancy 7.9 (6.2) ^(a) 4.1 (4.2) ^(a)Completeness, % 99.9 (100.0) ^(a) 99.7 (99.6) ^(a) R_(sym) 0.19 (0.79)^(a) 0.06 (0.57) ^(a) Mean I/σ (I) 8.1 (1.4) ^(a) 14.5 (3.0) ^(a) ^(a)Values in parentheses correspond to the highest resolution shell

CD40L formed a trimer (polypeptides A, B, and C in FIG. 17A). Each 309Tn3 scaffold (polypeptides D, E, and F in FIG. 17A) made contact withtwo CD40L polypeptides. The crystal structure revealed that there aresix specific contacts between each 309 scaffold and the first and secondCD40L polypeptides. Aspartic acid 17 in the BC makes contact withthreonine 251 in the first CD40L. Glutamic acid 18 in the BC loop makescontact with arginine 203 in the first CD40L and with isoleucine 204 inthe second CD40L. Serine 47 in the DE loop makes contact with histidine249 in the first CD40L. Tryptophan 49 in the DE loop makes contact withvaline 247 in the first CD40L. Aspartic acid 70 in the FG loop makescontact with serine 185 in the second CD40L (see FIG. 17A). CD40L aminoacid residues contacting the 311 scaffold are also shown in FIG. 18A.

An in the case of 309, each 311K4E_12 monomer scaffold (polypeptides A,B, and C in FIG. 17B) makes contact with two CD40L polypeptides. Thecrystal structure revealed that there are 19 specific contacts betweeneach 311K4E_12 scaffold and the first and second CD40L polypeptides.Asparagine 17 in the BC loop makes contacts with tyrosine 146 andglutamic acid 142 in the first CD40L. Arginine 18 in the BC loop makescontact with glutamic acid 142, tyrosine 146, and methionine 148 in thefirst CD40L. Serine 19 in the BC loop makes contact with glutamic acid142 and leucine 155 in the first CD40L. Serine 22 in the BC loop makescontact with asparagine 151 in the first CD40L. Histidine 15 in the BCloop makes contact with tyrosine 146 in the first CD40L. Histidine 51 inthe DE loop makes contact with tyrosine 146 in the first CD40L and withglutamic acid 230 in the second CD40L. Valine 50 in the DE loop makescontact with glutamic acid 230 in the second CD40L. The N-terminalregion of the 311K4E_12 monomer scaffold is connected to the secondCD40L. Arginine 200 in the second CD40L makes contact with threonine 7,aspartic acid 8, and threonine 10 in the N-terminal region of 311K4E_12.Arginine 203 in the second CD40L makes contact with glutamic acid 4 andaspartic acid 5. CD40L amino acid residues contacting the 309 scaffoldare also shown in FIG. 18B.

The crystal structures of CD40L in complexes with 309 and 311K4E_12showed that the 311K4E_12 and 309 monomer scaffolds bind to differentepitopes located in different parts of the CD40L trimer complex (FIG.17C). The structures showed that both scaffolds bind in the same groovethat would interact with the CD40 receptor.

The crystal structure of a 342 with CD40L is provided in FIG. 20 andshows that while 342 binds on the same part of CD40L specific changes inthe contact residues are seen as compared to the parental 309 clone.Specifically, in 342 aspartate 18 in the BC loop makes contact withthreonine 251 of CD40L and histidine 47 of the DE loop makes contactwith histidine 249 of CD40L, histidine 48 of the DE loop makes contactwith histidine 249, serine 245 and serine 248 of CD40L, and histidine 50of the DE loop makes contact with valine 247 of CD40L.

The crystal structure of a 342 and 311K4E_12 with CD40L demonstratesthat both scaffolds can bind simultaneously to their respective epitopeswhich are located in different parts of the CD40L trimer complex (FIG.21 ). The contacts for each of the separate scaffolds (as describedabove) are maintained.

The examples shown above illustrate various aspects of the invention andpractice of the methods of the invention. These examples are notintended to provide an exhaustive description of the many differentembodiments of the invention. Thus, although the invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, those of ordinary skill in the art willrealize readily that many changes and modifications can be made withoutdeparting from the spirit or scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

SEQUENCES CD40L sp|P29965|CD40L_HUMAN-Membrane formCytoplasmic domain = 1-20Signal anchor type II membrane protein region = 21-46Soluble form = 113-261 SEQ ID NO: 1MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLCD40L-Soluble form, corresponds also to the co-crystallized constructSEQ ID NO: 2MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLTn3 (with unmodified loops) SEQ ID No: 3IEVKDVTDTTALITWFKPLAEIDGCELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT 3^(rd) FnIII of tenascin C, AB loop (Tn3) SEQ ID NO: 4 KDVTDTT3^(rd) FnIII of tenascin C, BC loop (Tn3) SEQ ID NO: 5 FKPLAEIDG3^(rd) FnIII of tenascin C, CD loop (Tn3) SEQ ID NO: 6 KDVPGDR3^(rd) FnIII of tenascin C, DE loop (Tn3) SEQ ID NO: 7 TEDENQ3^(rd) FnIII of tenascin C, EF loop (Tn3) SEQ ID NO: 8 GNLKPDTE3^(rd) FnIII of tenascin C, FG loop (Tn3); also in 309FGwt, 340, 341, 342, 343,344, 345, 346, 347, 348, and 349 clones SEQ ID NO: 9 RRGDMSSNPA3^(rd) FnIII of tenascin C, beta strand A (Tn3) SEQ ID NO: 10 RLDAPSQIEV3^(rd) FnIII of tenascin C, beta strand A (Tn3) N-terminal truncationSEQ ID NO: 11 IEV 3^(rd) FnIII of tenascin C, beta strand B (Tn3)SEQ ID NO: 12 ALITW3^(rd) FnIII of tenascin C, beta strand C (Tn3 variant) SEQ ID NO: 13CELAYGI 3^(rd) FnIII of tenascin C, beta strand C (Tn3) SEQ ID NO: 14CELTYGI 3^(rd) FnIII of tenascin C, beta strand D (Tn3) SEQ ID NO: 15TTIDL 3^(rd) FnIII of tenascin C, beta strand E (Tn3) SEQ ID NO: 16 YSI3^(rd) FnIII of tenascin C, beta strand F (Tn3) SEQ ID NO: 17 YEVSLIC3^(rd) FnIII of tenascin C, beta strand G (Tn3) SEQ ID NO: 18 KETFTTClone 309-Parental clone isolated from naiive Tn3 library SEQ ID NO: 19AIEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICYTDQRAGNPAKETFTTGGGTLGHHHHHHHHClone 309-Parental clone isolated from naiive Tn3 library (w/o N-term A, andC-term linker and His8 tag) SEQ ID NO: 20IEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICYTDQEAGNPAKETFTT Clone 309FGwt-Parental clone with “humanized” FG loop  SEQ ID NO: 21AIEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 309FGwt-Parental clone with “humanized” FG loop (w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 22IEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 340-Affinity Mature variant SEQ ID NO: 23AIEVKDVTDTTALITWSDDFDNYEWCELTYGIKDVPGDRTTIDLWYHMAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 340-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 24IEVKDVTDTTALITWSDDFDNYEWCELTYGIKDVPGDRTTIDLWYHMAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 341-Affinity Mature variant SEQ ID NO: 25AIEVKDVTDTTALITWSDDFADYVWCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 341-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 26IEVKDVTDTTALITWSDDFADYVWCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 342-Affinity Mature variant (w/WT FG loop) SEQ ID NO: 27AIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 342-Affinity Mature variant (w/WT FG loop; w/o N-term A, and C-termlinker and His8 tag) SEQ ID NO: 28IEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 343-Affinity Mature variant SEQ ID NO: 29AIEVKDVTDTTALITWLDDWGSYHVCELTYGIKDVPGDRTTIDLWYHQAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 343-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 30IEVKDVTDTTALITWLDDWGSYHVCELTYGIKDVPGDRTTIDLWYHQAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 344-Affinity Mature variant SEQ ID NO: 31AIEVKDVTDTTALITWSDEVGDYVVCELTYGIKDVPGDRTTIDLWYHMAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 344-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 32IEVKDVTDTTALITWSDEVGDYVVCELTYGIKDVPGDRTTIDLWYHMAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 345-Affinity Mature variant SEQ ID NO: 33AIEVKDVTDTTALITWSDDFAEYVGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 345-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 34IEVKDVTDTTALITWSDDFAEYVGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 346-Affinity Mature variant SEQ ID NO: 35AIEVKDVTDTTALITWSDDFEEYVVCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 346-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 36IEVKDVTDTTALITWSDDFEEYVVCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 347-Affinity Mature variant SEQ ID NO: 37AIEVKDVTDTTALITWSDEVGQYVGCELTYGIKDVPGDRTTIDLWYHMAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 347-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 38IEVKDVTDTTALITWSDEVGQYVGCELTYGIKDVPGDRTTIDLWYHMAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 348-Affinity Mature variant SEQ ID NO: 39AIEVKDVTDTTALITWSDDIGLYVWCELTYGIKDVPGDRTTIDLWFHQAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 348-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 40IEVKDVTDTTALITWSDDIGLYVWCELTYGIKDVPGDRTTIDLWFHQAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 349-Affinity Mature variant SEQ ID NO: 41AIEVKDVTDTTALITWSDEHAEFIGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTTGGGTLGHHHHHHHHClone 349-Affinity Mature variant (w/o N-term A, and C-term linker and His8 tag)SEQ ID NO: 42IEVKDVTDTTALITWSDEHAEFIGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT Clone 311-Parental clone isolated from naiive Tn3 librarySEQ ID NO: 43AIEVKDVTDTTALITWTNRSSYYNLHGCELTYGIKDVPGDRTTIDLSSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311-Parental clone isolated from naiive Tn3 library(w/o N-term A, andC-term linker and His8 tag) SEQ ID NO: 44IEVKDVTDTTALITWTNRSSYYNLHGCELTYGIKDVPGDRTTIDLSSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTT Clone 311K4E-Variant from first round of affinity maturationSEQ ID NO: 45AIEVEDVTDTTALITWTNRSSYYNLHGCELTYGIKDVPGDRTTIDLSSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E-Variant from first round of affinity maturation(w/o N-term A, andC-term linker and His8 tag) SEQ ID NO: 46IEVEDVTDTTALITWTNRSSYYNLHGCELTYGIKDVPGDRTTIDLSSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_1-Clone Variant from second round of affinity maturationSEQ ID NO: 47AIEVEDVTDTTALITWINRSYYADLHGCELTYGIKDVPGDRTTIDLDQIYVHYSIGNLKPDTKYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_1-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 48IEVEDVTDTTALITWINRSYYADLHGCELTYGIKDVPGDRTTIDLDQIYVHYSIGNLKPDTKYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_2-Clone Variant from second round of affinity maturationSEQ ID NO: 49AIEVEDVTDTTALITWTNRSSYSHLDGCELTYGIKDVPGDRTTIDLSAAIYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_2-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 50IEVEDVTDTTALITWTNRSSYSHLDGCELTYGIKDVPGDRTTIDLSAAIYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_3-Clone Variant from second round of affinity maturationSEQ ID NO: 51AIEVEDVTDTTALITWINRSSYHNFPHCELAYGIKDVPGDRTTIDLNSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_3-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 52IEVEDVTDTTALITWINRSSYHNFPHCELAYGIKDVPGDRTTIDLNSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_4-Clone Variant from second round of affinity maturationSEQ ID NO: 53AIEVEDVTDTTALITWTNRSSYSNHLGCELAYGIKDVPGDRTTIDLNNIYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_4-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 54IEVEDVTDTTALITWTNRSSYSNHLGCELAYGIKDVPGDRTTIDLNNIYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_5-Clone Variant from second round of affinity maturationSEQ ID NO: 55AIEVEDVTDTTALITWTNRSSYSNFHGCELAYGIKDVPGDRTTIDLNSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_5-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 56IEVEDVTDTTALITWTNRSSYSNFHGCELAYGIKDVPGDRTTIDLNSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_7-Clone Variant from second round of affinity maturationSEQ ID NO: 57AIEVEDVTDTTALITWTNRSFYSNLHGCELTYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_7-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 58IEVEDVTDTTALITWTNRSFYSNLHGCELTYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_8-Clone Variant from second round of affinity maturationSEQ ID NO: 59AIEVEDVTDTTALITWTNRSSYAYLHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_8-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 60IEVEDVTDTTALITWTNRSSYAYLHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_9-Clone Variant from second round of affinity maturationSEQ ID NO: 61AIEVEDVTDTTALITWINRSSYANLHGCELTYGIKDVPGDRTTIDLSSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_9-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 62IEVEDVTDTTALITWINRSSYANLHGCELTYGIKDVPGDRTTIDLSSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_10-Clone Variant from second round of affinity maturationSEQ ID NO: 63AIEVEDVTDTTALITWTNRSSYANYHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_10-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 64IEVEDVTDTTALITWTNRSSYANYHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_11-Clone Variant from second round of affinity maturationSEQ ID NO: 65AIEVEDVTDTTALITWTNRSSYANLPGCELTYGIKDVPGDRTTIDLNSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_11-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 66IEVEDVTDTTALITWTNRSSYANLPGCELTYGIKDVPGDRTTIDLNSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_12-Clone Variant from second round of affinity maturationSEQ ID NO: 67AIEVEDVTDTTALITWTNRSSYSNLHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYNNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_12-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 68IEVEDVTDTTALITWTNRSSYSNLHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYNNPAKETFTTClone 311K4E_13-Clone Variant from second round of affinity maturationSEQ ID NO: 69AIEVEDVTDTTALITWINRSSYANLHGCELTYGIKDVPGDRTTIDLNSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_13-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 70IEVEDVTDTTALITWINRSSYANLHGCELTYGIKDVPGDRTTIDLNSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_14-Clone Variant from second round of affinity maturationSEQ ID NO: 71AIEVEDVTDTTALITWTARSAYSHHHYCELTYGIKDVPGDRTTIDLRQPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_14-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 72IEVEDVTDTTALITWTARSAYSHHHYCELTYGIKDVPGDRTTIDLRQPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_15-Clone Variant from second round of affinity maturationSEQ ID NO: 73AIEVEDVTDTTALITWTNRSSYANYHHCELTYGIKDVPGDRTTIDLELYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_15-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 74IEVEDVTDTTALITWTNRSSYANYHHCELTYGIKDVPGDRTTIDLELYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_16-Clone Variant from second round of affinity maturationSEQ ID NO: 75AIEVEDVTDTTALITWTNRSSYSDLPGCELTYGIKDVPGDRTTIDLSSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_16-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 76IEVEDVTDTTALITWTNRSSYSDLPGCELTYGIKDVPGDRTTIDLSSPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_19-Clone Variant from second round of affinity maturationSEQ ID NO: 77AIEVEDVTDTTALITWTHRSAYSNHSFCELTYGIKDVPGDRTTIDLNTPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_19-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 78IEVEDVTDTTALITWTHRSAYSNHSFCELTYGIKDVPGDRTTIDLNTPYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_20-Clone Variant from second round of affinity maturationSEQ ID NO: 79AIEVEDVTDTTALITWTNRSLYANFHGCELTYGIKDVPGDRTTIDLEQVYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_20-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 80IEVEDVTDTTALITWTNRSLYANFHGCELTYGIKDVPGDRTTIDLEQVYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTClone 311K4E_21-Clone Variant from second round of affinity maturationSEQ ID NO: 81AIEVEDVTDTTALITWTNRSSYSNLPGCELTYGIKDVPGDRTTIDLNQVYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTTGGGTLGHHHHHHHHClone 311K4E_21-Clone Variant from second round of affinity maturation(w/o N-term A, and C-term linker and His8 tag) SEQ ID NO: 82IEVEDVTDTTALITWTNRSSYSNLPGCELTYGIKDVPGDRTTIDLNQVYVHYSIGNLKPDTEYEVSLICLTTDGTYSNPAKETFTT Clone 309 and 309FGwt-BC Loop SEQ ID NO: 83 SDEFGHYDGClone 340-BC Loop SEQ ID NO: 84 SDDFDNYEW Clone 341-BC LoopSEQ ID NO: 85 SDDFADYVW Clone 342-BC Loop SEQ ID NO: 86 SDDFGEYVWClone 343-BC Loop SEQ ID NO: 87 LDDWGSYHV Clone 344-BC LoopSEQ ID NO: 88 SDEVGDYVV Clone 345-BC Loop SEQ ID NO: 89 SDDFAEYVGClone 346-BC Loop SEQ ID NO: 90 SDDFEEYVV Clone 347-BC LoopSEQ ID NO: 91 SDEVGQYVG Clone 348-BC Loop SEQ ID NO: 92 SDDIGLYVWClone 349-BC Loop SEQ ID NO: 93 SDEHAEFIGClone 309, 309FGwt, 341, 345, 346, 349-DE Loop SEQ ID NO: 94 WWHSAWClone 340, 344, 347-DE Loop SEQ ID NO: 95 WYHMAW Clone 342-DE LoopSEQ ID NO: 96 WYHHAH Clone 343-DE Loop SEQ ID NO: 97 WYHQAWClone 348-DE Loop SEQ ID NO: 98 WFHQAW Clone 309-FG Loop SEQ ID NO: 99YTDQEAGNPA Clone 311, 311K4E-BC Loop SEQ ID NO: 100 TNRSSYYNLHGClone 311K4E_1-BC Loop SEQ ID NO: 101 INRSYYADLHG Clone 311K4E_2-BC LoopSEQ ID NO: 102 TNRSSYSHLDG Clone 311K4E_3-BC Loop SEQ ID NO: 103INRSSYHNFPH Clone 311K4E_4-BC Loop SEQ ID NO: 104 TNRSSYSNHLGClone 311K4E_5-BC Loop SEQ ID NO: 105 TNRSSYSNFHG Clone 311K4E_7-BC LoopSEQ ID NO: 106 TNRSFYSNLHG Clone 311K4E_8-BC Loop SEQ ID NO: 107TNRSSYAYLHG Clone 311K4E_9, 311K4E_13-BC Loop SEQ ID NO: 108 INRSSYANLHGClone 311K4E_10-BC Loop SEQ ID NO: 109 TNRSSYANYHGClone 311K4E_11-BC Loop SEQ ID NO: 110 TNRSSYANLPGClone 311K4E_12-BC Loop SEQ ID NO: 111 TNRSSYSNLHGClone 311K4E_14-BC Loop SEQ ID NO: 112 TARSAYSHHHYClone 311K4E_15-BC Loop SEQ ID NO: 113 TNRSSYANYHHClone 311K4E_16-BC Loop SEQ ID NO: 114 TNRSSYSDLPGClone 311K4E_19-BC Loop SEQ ID NO: 115 THRSAYSNHSFClone 311K4E_20-BC Loop SEQ ID NO: 116 TNRSLYANFHGClone 311K4E_21-BC Loop SEQ ID NO: 117 TNRSSYSNLPGClone 311, 311K4E, 311K4E_9, 311K4E_16-DE Loop SEQ ID NO: 118 SSPYVHClone 311K4E_1-DE Loop SEQ ID NO: 119 DQIYVH Clone 311K4E_2-DE LoopSEQ ID NO: 120 SAAIYVHClone 311K4E_3, 311K4E_5, 311K4E_11, 311K4E_13-DE Loop SEQ ID NO: 121NSPYVH Clone 311K4E_4-DE Loop SEQ ID NO: 122 NNIYVHClone 311K4E_7, 311K4E_8, 311K4E_10, 311K4E_12-DE Loop SEQ ID NO: 123NQPYVH Clone 311K4E_14-DE Loop SEQ ID NO: 124 RQPYVHClone 311K4E_15-DE Loop SEQ ID NO: 125 ELYVH Clone 311K4E_19-DE LoopSEQ ID NO: 126 NTPYVH Clone 311K4E_20-DE Loop SEQ ID NO: 127 EQVYVHClone 311K4E_21-DE Loop SEQ ID NO: 128 NQVYVHClone 311, 311K4E, 311K4E_1, 311K4E_2, 311K4E_3, 311K4E_4, 311K4E_5,311K4E_7, 311K4E_8, 311K4E_9, 311K4E_10, 311K4E_11, 311K4E_13, 311K4E_14,311K4E_15, 311K4E_16, 311K4E_19, 311K4E_20, 311K4E_21-FG LoopSEQ ID NO: 129 LTTDGTYSNPA Clone 311K4E_12-FG Loop SEQ ID NO: 130LTTDGTYNNPA 2GS Linker-(Gly4Ser)2 SEQ ID NO: 131 GGGGSGGGGS3GS Linker-(Gly4Ser)3 SEQ ID NO: 132 GGGGSGGGGSGGGGS HSA C34S mutantCys->Ser mutation location is underlined SEQ ID NO: 133DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADEVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 342-2GS-HSAC34S-Monovalent ConstructHSA C34S is underlined SEQ ID NO: 134SQIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADEVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL342-3GS-342-2GS-HSAC34S Bivalent Construct HSA is underlinedSEQ ID NO: 135SQIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGGGGSGGGGSGGGGSRLDAPSQIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADEVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL311 clone family AB loop N-term E variant SEQ ID NO: 136 EDVTDTT311 clone family EF loop C-term K variant SEQ ID NO: 137 GNLKPDTKHSA human full-length SEQ ID NO: 138DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADEVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL309 FG loop variant (RR -> RS mutant); may be present in 342 constructsSEQ ID NO: 139 RSGDMSSNPA2GX Linker-(Gly4X)2; X = Ala, Gly, Leu, Ile, Val SEQ ID NO: 140GGGGXGGGGX 3GX Linker-(Gly4X)3; X = Ala, Gly, Leu, Ile, ValSEQ ID NO: 141 GGGGXGGGGXGGGGX G10 Linker-(Gly4Gly)2 SEQ ID NO: 142GGGGGGGGGG G15Linker-(Gly4Gly)3 SEQ ID NO: 143 GGGGGGGGGGGGGGG342-G10-HSAC34S-Monovalent construct 2-all Gly linkers HSA is underlinedSEQ ID NO: 144SQIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGGGGGGGGGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL342-G15-342-G10-HSAC34S Bivalent construct 2-all Gly linkersHSA is underlined SEQ ID NO: 145SQIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGGGGGGGGGGGGGGGRLDAPSQIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGGGGGGGGGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLClone 342-Affinity Mature variant (w/FG loop variant RR->RS underlined)SEQ ID NO: 146IEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGly-Ser linker module, (G4S)_(n) where n = 1-7; the (G₄S)_(n) module wherein n = 1 isshown SEQ ID NO: 147 GGGGS Gly linker module (G₅)_(n) where n = 1-7; the (G₅)_(n) module wherein n = 1 is shownSEQ ID NO: 148 GGGGGGly-Ala linker module, (G4A)n where n = 1-7; the (G₄A)_(n) module wherein n = 1is shown SEQ ID NO: 149 GGGGA Poly-Histidine Tag (H₈)-An optional component of the Tn3 scaffolds usefulfor purification maybe combined with additional linker residues.SEQ ID NO: 150 HHHHHHHHLinker-Poly-Histidine Tag-An optional component of the Tn3 scaffoldsuseful for purification SEQ ID NO: 151 GGGGSHHHHHHHHMature MSA wild type SEQ ID NO: 152EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADEVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALAMature MSA-C34S/C579S Cys mutant; mutated residues underlinedSEQ ID NO: 153EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKSSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADEVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRSKDALA Clone M13; BC, DE, FG loops are underlinedSEQ ID NO: 154IEVKDVTDTTALITWHDAFGYDFGCELTYGIKDVPGDRTTIDLPDHFHNYSIGNLKPDTEYEVSLICANDHGFDSNPAKETFTT Clone M13N49Q; N49Q mutation underlined SEQ ID NO: 155IEVKDVTDTTALITWHDAFGYDFGCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSNPAKETFTT M13N49Q-1GS-M13N49Q bivalent Construct; N49Q mutation underlinedSEQ ID NO: 156SQIEVKDVTDTTALITWHDAFGYDFGCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSNPAKETFTTTGGGGSRLDAPSQIEVKDVTDTTALITWHDAFGYDFGCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSNPAKETFTTM13N49Q-3GS-MSA-C34S/C579S monovalent construct; mutations underlinedSEQ ID NO: 157SQIEVKDVTDTTALITWHDAFGYDFGCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSNPAKETFTTGGGGSGGGGSGGGGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKSSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADEVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRSKDALAM13N49Q-1GS-M13N49Q-3GS-MSA-C34S/C579S bivalent construct; mutations underlinedSEQ ID NO: 158SQIEVKDVTDTTALITWHDAFGYDFGCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSNPAKETFTTTGGGGSRLDAPSQIEVKDVTDTTALITWHDAFGYDFGCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSNPAKETFTTGGGGSGGGGSGGGGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKSSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADEVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRSKDALAClone M31; BC, DE, FG loops are underlined SEQ ID NO: 159IEVKDVTDTTALITWHDPSGYDFWCELTYGIKDVPGDRTTIDLPDHFHNYSIGNLKPDTEYEVSLICANDHGFDSYPAKETFTT Clone M31N49Q; N49Q mutation underlined SEQ ID NO: 160IEVKDVTDTTALITWHDPSGYDFWCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSYPAKETFTT M31N49Q-1GS-M31N49Q bivalent construct; N49Q mutation underlinedSEQ ID NO: 161SQIEVKDVTDTTALITWHDPSGYDFWCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSYPAKETFTTTGGGGSRLDAPSQIEVKDVTDTTALITWHDPSGYDFWCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSYPAKETFTTM31N49Q-3GS-MSA-C34S/C579S monovalent construct; mutations underlinedSEQ ID NO: 162SQIEVKDVTDTTALITWHDPSGYDFWCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSYPAKETFTTGGGGSGGGGSGGGGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKSSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADEVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRSKDALAM31N49Q-1G5-M31N49Q-3GS-MSA-C34S/C579S bivalent construct; mutations underlinedSEQ ID NO: 163SQIEVKDVTDTTALITWHDPSGYDFWCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSYPAKETFTTTGGGGSRLDAPSQIEVKDVTDTTALITWHDPSGYDFWCELTYGIKDVPGDRTTIDLPDHFHQYSIGNLKPDTEYEVSLICANDHGFDSYPAKETFTTGGGGSGGGGSGGGGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKSSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADEVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRSKDALAClone D1-Negative control Tn3 SEQ ID NO: 164IEVKDVTDTTALITWSPGERIWMFTGCELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSLICPNYERISNPAKETFTTTD1-1GS-D1-3G-MSA-C34S/C579S bivalent construct; mutations underlinedSEQ ID NO: 165SQIEVKDVTDTTALITWSPGERIWMFTGCELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSLICPNYERISNPAKETFTTTGGGGSRLDAPSQIEVKDVTDTTALITWSPGERIWMFTGCELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSLICPNYERISNPAKETFTTGGGGSGGGGSGGGGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKSSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRSKDALAClone 342 RDG to SDG mutant; mutation underlined SEQ ID NO: 166IEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTT 309FGwt consensusAll strands are parent Tn3 strands; beta strand C is CELTYGI variant (SEQ IDNO: 14); AB, CD, EF loop are parent Tn3 loops X1 = Ser or LeuX2 = Asp or Glu X3 = His, Ile, Val, Phe or Trp X4 = Ala, Gly, Glu or AspX5 = Glu, Leu, Gln, Ser, Asp or Asn X6 = Phe or TyrX7 = Ile, Val, His, Glu or Asp X8 = Gly, Trp or Val X9 = Trp, Phe or TyrX10 = Ser, Gln, Met or His X11 = Trp or His X12 = Arg or SerSEQ ID NO: 167IEVKDVTDTTALITWX1DX2X3X4X5X6X7X8CELTYGIKDVPGDRTTIDLWX9HX10AX11YSIGNLKPDTEYEVSLICRX12GDMSSNPAKETFTT 309FGwt consensus, BC loop  X1 = Ser or LeuX2 = Asp or Glu X3 = His, Ile, Val, Phe or Trp X4 = Ala, Gly, Glu or AspX5 = Glu, Leu, Gln, Ser, Asp or Asn X6 = Phe or TyrX7 = Ile, Val, His, Glu or Asp X8 = Gly, Trp or Val SEQ ID NO: 168X1DX2X3X4X5X6X7X8 309FGwt consensus, DE loop  X9 = Trp, Phe or TyrX10 = Ser, Gln, Met or His X11 = Trp or His SEQ ID NO: 169 WX9HX10AX11309FGwt consensus, FG loop  X12 = Arg or Ser SEQ ID NO: 170 RX12GDMSSNPA311 consensus; all strands are parent Tn3 strands; two beta strand C variants(SEQ ID NO: 13 and 14); CD loop is parent Tn3 loop  X1 = Lys or GluX2 = Thr or Ile X3 = Asn or Ala X4 = Ser, Leu, Ala, Phe or TyrX5 = Tyr, Ala, Gly, Val, Ile or Ser (BC/N-term contact)X6 = Tyr, Ser, Ala or His X7 = Asn, Asp, His or TyrX8 = Leu, Phe, His or Tyr X9 = His, Pro, Ser, Leu or AspX10 = Gly, Phe, His or Tyr X11 = Ala or ThrX12 = Ser, Asn, Glu, Arg or Asp X13 = Ser, Gln, Thr, Asn or AlaX14 = Pro, Val, -, Ile or Ala (- no amino acid)X15 = - or Ile (- no amino acid) X16 = Glu or Lys X17 = Ser or AsnSEQ ID NO: 171IEVX1DVTDTTALITWX2X3RSX4X5X6X7X8X9X1OCELX11YGIKDVPGDRTTIDLX12X13X14X15YVHYSIGNLKPDTX16YEVSLICLTTDGTYX17NPAKETFTT311 consensus; beta strand C in 311 family clones X11 = Ala or ThrSEQ ID NO: 172 CELX11YGI 311 consensus; AB loop  X1 = Lys or GluSEQ ID NO: 173 X1DVTDTT 311 consensus; BC loop  X2 = Thr or IleX3 = Asn or AlaX4 = Ser, Leu, Ala, Phe or Tyr X5 = Tyr, Ala, Gly, Val, Ile or Ser (BC/N-termcontact) X6 = Tyr, Ser, Ala or His X7 = Asn, Asp, His or TyrX8 = Leu, Phe, His or Tyr X9 = His, Pro, Ser, Leu or AspX10 = Gly, Phe, His or Tyr SEQ ID NO: 174 X2X3RSX4X5X6X7X8X9X10311 consensus; DE loop X12 = Ser, Asn, Glu, Arg or Asp X13 = Ser, Gln, Thr, Asn or AlaX14 = Pro, Val, -, Ile or Ala (- no amino acid)X15 = - or Ile (- no amino acid) SEQ ID NO: 175 X12X13X14X15YVH311 consensus; EF loop  X16 = Glu or Lys SEQ ID NO: 176 GNLKPDTX16311 consensus; FG loop  X17 = Ser or Asn SEQ ID NO: 177 LTTDGTYX17NPABC9 NHT oligo; loop BCNucleotide codes: N = G/A/T/C; H = A/T/C; R = A/G; S = G/C; B = T/C/G;V = A/C/G; M = A/C; K = G/T SEQ ID NO: 178ACCGCGCTGATTACCTGGNHTNHTSCGNHTGSTNHTNHTNHTGGCTGTGAACTGACCTAT GGCATTAAABC11 NHT oligo; loop BCNucleotide codes: N = G/A/T/C; H = A/T/C; R = A/G; S = G/C; B = T/C/G;V = A/C/G; M = A/C; K = G/T SEQ ID NO: 179ACCGCGCTGATTACCTGGNHTNHTBSTNHTNHTNHTNHTNHTNHTNHTGGCTGTGAACTGACCTATGGCATTAAABC12 NHT oligo; loop BCNucleotide codes: N = G/A/T/C; H = A/T/C; R = A/G; S = G/C; B = T/C/G;V = A/C/G; M = A/C; K = G/T SEQ ID NO: 180ACCGCGCTGATTACCTGGNHTVMACCGNHTNHTNHTRRCRGCNHTVTTNHTGGCTGTGAACTGACCTATGGCATTAAADE NHT oligo; DE loop Nucleotide codes: N = G/A/T/C; H = A/T/C; R = A/G; S = G/C; B = T/C/G;V = A/C/G; M = A/C; K = G/T SEQ ID NO: 181CGATCGCACCACCATAGATCTGNHTNHTNHTNHTNHTNHTTATAGCATTGGTAACCTGAAACCGFG9 NHT oligo; FG loop Nucleotide codes: N = G/A/T/C; H = A/T/C; R = A/G; S = G/C; B = T/C/G;V = A/C/G; M = A/C; K = G/T SEQ ID NO: 182GAATATGAAGTGAGCCTGATTTGCNHTAMSNHTNHTGGTNHTNHTNHTKCGAAAGAAACCTTTACCACCGGTGFG10 NHT oligo; FG loop Nucleotide codes: N = G/A/T/C; H = A/T/C; R = A/G; S = G/C; B = T/C/G;V = A/C/G; M = A/C; K = G/T SEQ ID NO: 183GAATATGAAGTGAGCCTGATTTGCNHTAMSNHTNHTNHTNHTRGCNHTCCGGCGAAAGAAACCTTTACCACCGGTGFG11 NHT oligo; FG loopNucleotide codes: N = G/A/T/C; H = A/T/C; R = A/G; S = G/C; B = T/C/G;V = A/C/G; M = A/C; K = G/T SEQ ID NO: 184GAATATGAAGTGAGCCTGATTTGCNHTAMSNHTNHTGGTNHTNHTAGCAACCCGGCGAAAGAAACCTTTACCACCGGTGBCX-DE bridge v2 oligo SEQ ID NO: 185CAGATCTATGGTGGTGCGATCGCCCGGCACATCTTTAATGCCATAGGTCAGTTCACADE-FGX bridge v2 oligo SEQ ID NO: 186GCAAATCAGGCTCACTTCATATTCGGTATCCGGTTTCAGGTTACCAATGCTATKpnI amp rev v2 oligo SEQ ID NO: 187CGGGTCGGTTGGGGTACCGCCACCGGTGGTAAAGGTTTCTTT KpnI reverse v2 oligoSEQ ID NO: 188 CGGGTCGGTTGGGGTA BC library amp v2 oligo SEQ ID NO: 189GGCCCAGCCGGCCATGGCCGCCATTGAAGTGAAAGATGTGACCGATACCACCGCGCTGATTACCTGGBC9 PCR oligoNucleotide codes: 1 = codons for all 19aa(-cys); 2 = codons for Ala/Pro 50/50;3 = codons for Ala/Gly SEQ ID NO: 190ACCGCGCTGATTACCTGGTCT1213111GGCTGTGAACTGACCTATGGCATTAAAGATGBC 9-loop NNK oligo Nucleotide codes: K= 50%G/50%T SEQ ID NO: 191ACCGCGCTGATTACCTGGNNKNNKSMGNNKGSTNNKNNKNNKGGCTGTGAACTGACCTATGGCATTAAA309 BC-loop NNKdope oligoNucleotide codes: 4 = 70%G10%A10%C10%T; 5 = 10%G, 70%A, 10%C, 10%T;6 = 10%G, 10%A, 70%C, 10%T; 7 = 10%G, 10%A, 10%C, 70%T; 8 = 70%A15%C15%T; andK = 50%G/50%T SEQ ID NO: 192ACCGCGCTGATTACCTGG76K45K45K77K44K65K78T45K44KTGTGAACTGACCTATGGCATTAAADE PCR oligo Nucleotide codes: 1 = codons for all 19aa(-cys)SEQ ID NO: 193GATGTGCCGGGCGATCGCACCACCATAGATCTG111111TATAGCATTGGTAACCTGAAACCGGUpstr BCloop Rev oligo SEQ ID NO: 194 CCAGGTAATCAGCGCGGTGGTATBC shuffle rev oligo SEQ ID NO: 195 CAGATCTATGGTGGTGCGATCGCDE shuffle FWD oligo SEQ ID NO: 196 TGTGAACTGACCTATGGCATTAAAGATGTBC11-311Gly oligoNucleotide codes: 1 = 70%G, 10%A, 10%C, 10%T; 2 = 10%G, 70%A, 10%C, 10%T;3 = 10%G, 10%A, 70%C, 10%T; 4 = 10%G, 10%A, 10%C, 70%T; 5 = 70%A, 15%C, 15%T;6 = 15%A, 70%C, 15%T; 7 = 15%A, 15%C, 70%T; V = 33%A, 33%C, 33%G.SEQ ID NO: 197ACCGCGCTGATTACCTGG26T25TV1T46T46T45T45T25T37T35TGGCTGTGAACTGACCTATGGCATTAAABC11-311NHT oligoNucleotide codes: 1 = 70%G, 10%A, 10%C, 10%T; 2 = 10%G, 70%A, 10%C, 10%T;3 = 10%G, 10%A, 70%C, 10%T; 4 = 10%G, 10%A, 10%C, 70%T; 5 = 70%A, 15%C, 15%T;6 = 15%A, 70%C, 15%T; 7 = 15%A, 15%C, 70%T; V = 33%A, 33%C, 33%G; andH = 33%A, 33%C, 33%T SEQ ID NO: 198ACCGCGCTGATTACCTGG26T25TV1T46T46T45T45T25T37T35TNHTTGTGAACTGACCTATGGCATTAAABC library amp K4E oligo SEQ ID NO: 199GGCCCAGCCGGCCATGGCCGCCATTGAAGTGGAAGATGTGACCGATACCACCGCGCTGATTACCTGGExtended half-life HSA variant(C34S, L463N, K524L); mutations are underlinedSEQ ID NO: 200DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVNHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQILKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL311K4E_12-I variant monovalent construct (comprises GS linker and C34S HSA);linker and mutated serine are underlined SEQ ID NO: 201SQIEVEDVTDTTALITWTNRSSYSNLHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYNNPAKETFTTGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL311K4E_12-I variant monovalent construct (comprises beta strand C CELTYGvariant, all G linker, and C34S HSA); linker and mutated serine areunderlined SEQ ID NO: 202SQIEVEDVTDTTALITWTNRSSYSNLHGCELTYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYNNPAKETFTTGGGGGGGGGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL311K4E_12-I variant bivalent construct (comprises GS linkers and C34S HSA);linkers and mutated serine are underlined SEQ ID NO: 203SQIEVEDVTDTTALITWTNRSSYSNLHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYNNPAKETFTTGGGGSGGGGSGGGGSRLDAPSQIEVEDVTDTTALITWTNRSSYSNLHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYNNPAKETFTTGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL311K4E_12-I variant bivalent construct (comprises all G linkers and S34 HSA);linkers and mutated serine are underlined SEQ ID NO: 204SQIEVEDVTDTTALITWTNRSSYSNLHGCELTYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYNNPAKETFTT GGGGGGGGGGGGGGGRLDAPSQIEVEDVTDTTALITWTNRSSYSNLHGCELAYGIKDVPGDRTTIDLNQPYVHYSIGNLKPDTEYEVSLICLTTDGTYNNPAKETFTT GGGGGGGGGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL309-3GS-309 bivalent construct SEQ ID NO: 205SQIEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICYTDQEAGNPAKETFTTGGGGSGGGGSGGGGSRLDAPSQIEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICYTDQEAGNPAKETFTT 309-2GS-HSAC34S monovalent constructSEQ ID NO: 206SQIEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICYTDQEAGNPAKETFTTGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL309-3GS-309-2GS-HSAC34S bivalent construct SEQ ID NO: 207SQIEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICYTDQEAGNPAKETFTTGGGGSGGGGSGGGGSRLDAPSQIEVKDVTDTTALITWSDEFGHYDGCELTYGIKDVPGDRTTIDLWWHSAWYSIGNLKPDTEYEVSLICYTDQEAGNPAKETFTTGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL342-3GS-342 bivalent construct SEQ ID NO: 208SQIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGGGGSGGGGSGGGGSRLDAPSQIEVKDVTDTTALITWSDDFGEYVWCELTYGIKDVPGDRTTIDLWYHHAHYSIGNLKPDTEYEVSLICRSGDMSSNPAKETFTTGly-Ser linker module, (G4X)_(n) where X = G, S, A, L, I, or V and n = 1-7; the(G₄X)_(n) module wherein n = 1 is shown SEQ ID NO: 209 GGGGXOppA signal peptide mutant L25/M SEQ ID NO: 210MTNITKRSLVAAGVLAALMAGNVAMA

1.-121. (canceled)
 122. A method of reducing binding of CD40 ligand(CD40L) to CD40 receptor comprising administering an effective amount ofa composition comprising a Tn3 scaffold to a subject in need, whereinthe Tn3 scaffold comprises at least one CD40L-specific monomer subunit,wherein the monomer subunit comprises seven beta strands designated A,B, C, D, E, F, and G, and six loop regions designated AB, BC, CD, DE,EF, and FG, wherein the AB loop comprises SEQ ID NO: 4, the BC loopcomprises a sequence selected from the group consisting of SEQ ID NOS:83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 168, the CD loopcomprises SEQ ID NO: 6, the DE loop comprises a sequence selected fromthe group consisting of SEQ ID NOs: 94, 95, 96, 97, 98, and 169, the EFloop comprises SEQ ID NO: 8, and the FG loop comprises a sequenceselected from the group consisting of SEQ ID NOS: 9, 99, 139, and 170.123. The method of claim 122, wherein the CD40L-specific monomer subunitcomprises seven beta strands designated A, B, C, D, E, F, and G, whereinthe A beta strand consists of SEQ IID NO:11, the B beta strand consistsof SEQ ID NO:12, the C beta strand consists of SEQ ID NO:13 or SEQ IDNO:14, the D beta strand consists of SEQ ID NO:15, the E beta strandconsists of SEQ ID NO:16, the F beta strand consists of SEQ ID NO:17,and the G beta strand consists of SEQ ID NO:18, and six loop regionsdesignated AB, BC, CD, DE, EF, and FG.
 124. The method of claim 122,wherein the AB loop consists of SEQ ID NO: 4, the BC loop consists ofSEQ ID NO: 86, the CD loop consists of SEQ ID NO: 6, the DE loopconsists of SEQ ID NO: 96, the EF loop consists of SEQ ID NO: 8, and theFG loop consists of SEQ ID NO:
 139. 125. The method of claim 122,wherein the CD40L-specific monomer subunit comprises SEQ ID NO: 146.126. The method of claim 122, wherein the Tn3 scaffold comprises SEQ IDNO:
 145. 127. The method of claim 122, wherein the subject in need hascancer, an autoimmune disease, an infectious disease, or an inflammatorydisease.
 128. The method of claim 127, wherein the subject in need hasan autoimmune disease, and the autoimmune disease is selected from thegroup consisting of: alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunediseases of the adrenal gland, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome,atherosclerosis, a retinopathy, retrolental fibroplasia, age-relatedmacular degeneration, neovascular glaucoma, hemangiomas, Grave'sdisease, sepsis, arthritis, peritonitis, Crohn's disease, reperfusioninjury, septicemia, endotoxic shock, cystic fibrosis, endocarditis,psoriasis, anaphylactic shock, organ ischemia, spinal cord injury andallograft rejection, autoimmune thrombocytopenia, Behçet's disease,bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronicfatigue immune dysfunction syndrome (CFIDS), chronic inflammatorydemyelinating polyneuropathy, Churg-Strauss syndrome, cicatricialpemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-, glomerulonephritis,Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy,lichen planus, Meniere's disease, mixed connective tissue disease,multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis,pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, Raynaud's phenomenon, Reiter's syndrome, sarcoidosis,scleroderma, stiff-man syndrome, systemic lupus erythematosus, lupuserythematosus, Takayasu arteritis, temporal arteritis/giant cellarteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, andWegener's granulomatosis.
 129. The method of claim 128, wherein theautoimmune disease is Sjogren's syndrome.
 130. The method of claim 128,wherein the autoimmune disease is arthritis.
 131. The method of claim130, wherein the arthritis is juvenile arthritis.
 132. The method ofclaim 130, wherein the arthritis is rheumatoid arthritis.
 133. Themethod of claim 128, wherein the autoimmune disease is systemic lupuserythematosus.
 134. The method of claim 122, wherein the subject in needis a transplant recipient.
 135. The method of claim 134, wherein themethod is effective in reducing graft-vs-host disease in the subject.136. A treatment regimen, comprising administering to a subject in need:a) a Tn3 scaffold that comprises at least one CD40L-specific monomersubunit, wherein the monomer subunit comprises seven beta strandsdesignated A, B, C, D, E, F, and G, and six loop regions designated AB,BC, CD, DE, EF, and FG, wherein the AB loop comprises SEQ ID NO: 4, theBC loop comprises a sequence selected from the group consisting of SEQID NOS: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 168, the CD loopcomprises SEQ ID NO: 6, the DE loop comprises a sequence selected fromthe group consisting of SEQ ID NOs: 94, 95, 96, 97, 98, and 169, the EFloop comprises SEQ ID NO: 8, and the FG loop comprises a sequenceselected from the group consisting of SEQ ID NOS: 9, 99, 139, and 170;and b) a conventional therapy selected from the group consisting of:surgery, immunotherapy, chemotherapy, radiation therapy, and a smallmolecule.
 137. The method of claim 136, wherein the conventional therapyis administered concurrent with the Tn3 scaffold.
 138. The method ofclaim 137, wherein the conventional therapy is non-concurrentlyadministered.
 139. The method of claim 136, wherein the conventionaltherapy is surgery, and comprises a transplant.
 140. The method of claim136, wherein the conventional therapy is immunotherapy and comprises anantibody or portion thereof.
 141. The method of claim 136, wherein theCD40L-specific monomer subunit comprises SEQ ID NO:
 146. 142. The methodof claim 136, wherein the Tn3 scaffold comprises SEQ ID NO: 145.